Best was born as Jewel Jules Franklin Guy on July 26, 1926, in Powderly, Kentucky, to Lark and Lena Guy. His mother was the sister of Ike Everly, the father of the pop duo The Everly Brothers. After his mother died of tuberculosis in 1929, then three-year-old James was sent to live in an orphanage. He was later adopted by Armen Best (1897-1984) and his wife Essa (1896-1988) and went to live with them in Corydon, Indiana. He served honorably in the United States Army in World War II, training in 1944 in Biloxi, Mississippi as a gunner on a B-17 bomber, but by the time he finished training the war was almost at an end and he joined the Military Police. As an MP, Best played a role in bringing stability to war-torn Germany immediately after their surrender. While stationed in Germany, Best transferred out of the MP and joined a special services unit of actors for the US Army that travelled around Europe performing plays for the troops. This was the true beginning of his acting career.
Best began his screen acting career with an uncredited role in the 1950 film, One Way Street. Some of his more notable roles include Jason Brown in the 1955 historical drama about the abolitionist John Brown titled Seven Angry Men and as Kit Caswell in the 1958 western Cole Younger, Gunfighter, based on the infamous outlaw. He was further cast as Private Ridges in the 1958 film adaptation of Norman Mailer's The Naked and the Dead. He played the outlaw Billy John in Ride Lonesome (1959), Dr. Ben Mizer in the 1966 comedy Three on a Couch, the gunfighter Drew in Firecreek (1968), with James Stewart and Henry Fonda, and the cross-dressing Dewey Barksdale in the 1976 drama Ode to Billy Joe.
Best guest-starred more than 280 times in numerous television series. In 1954, he played the outlaw Dave Ridley, opposite Gloria Winters as the female bandit "Little Britches" in an episode of Stories of the Century. In 1954, Best appeared twice on the syndicated Annie Oakley series, starring Gail Davis and Brad Johnson. He was cast in the religion anthology series, Crossroads, in the 1956 episode "The White Carnation". He was also cast on an episode of Jackie Cooper's early NBC sitcom, The People's Choice and in the David Janssen crime drama, Richard Diamond, Private Detective.
In 1960, Best appeared in the episode "Love on Credit" of CBS's anthology series, The DuPont Show with June Allyson. He starred in Season 3, episode 7 of "The Twilight Zone" ("The Grave"). In 1963, he was cast as the courageous Wisconsin game warden, Ernie Swift, in the episode "Open Season" of another CBS anthology series, GE True, hosted by Jack Webb. In the story line, Swift faces the reprisal of organized crime after he tickets gangster Frank MacErlane (David McLean) for illegal fishing.
In 1962, he played the part of Art Fuller in the episode "Incident of El Toro" on CBS's Rawhide and in 1963, he returned to play Willie Cain in the episode "Incident at Spider Rock". Best made two guest appearances on Perry Mason. In 1963 he played title character Martin Potter in "The Case of the Surplus Suitor," and in 1966 he played defendant and oilman Allan Winford in "The Case of the Unwelcome Well". He appeared on a long list of other television series including Wagon Train (three times), The Adventures of Kit Carson (twice as Henry Jordan), the western anthology series Frontier (twice), The Rebel, Bonanza, Sheriff of Cochise, Pony Express, Rescue 8, Behind Closed Doors, The Texan, Gunsmoke, Have Gun – Will Travel, The Barbara Stanwyck Show, Tombstone Territory, Whispering Smith, Trackdown, The Rifleman, Stagecoach West, The Twilight Zone ("The Grave", "The Last Rites of Jeff Myrtlebank," and "Jess-Belle"), Wanted: Dead or Alive, Overland Trail, Bat Masterson, Alfred Hitchcock Presents, The Man and the Challenge, Combat!, The Green Hornet ("Deadline For Death"), The Mod Squad, I Spy, The Fugitive and In the Heat of the Night.
Best played Sheriff Rosco P. Coltrane on CBS's The Dukes of Hazzard from the debut of the program in 1979 until the series ended in 1985. This role was Best's most visible success. He later revealed that the caricature-like persona of Sheriff Coltrane was developed from a voice that he used when playing with his young children. On set, Best was particularly close to Sorrell Booke, who played the character of Boss Hogg, who was both the boss and the brother-in-law of Rosco. The two actors became close friends and according to interviews by the series creators, the two would often improvise their scenes together, making up their own dialogue as they went along. Until his death, he also remained close to Catherine Bach, who played the character of Daisy Duke, and long after the show's cancellation, she was a regular visitor to the website dedicated to Best's painting.
In 1991, in contrast to the comical Rosco Coltrane of Dukes of Hazzard, Best appeared in an episode of the NBC crime drama In the Heat of the Night. He won the Crystal Reel Award for Best Actor for his portrayal of Nathan Bedford in the episode "Sweet, Sweet Blues", directed by Vincent McEveety and written by William James Royce, Best plays a retired Sheriff and repentant killer who has to come to terms with being involved in the death of now Sparta police officer Sweet's grandfather.
He later moved to Florida and taught at the University of Central Florida (Orlando). After semi-retiring, Best ran a production company and took occasional acting roles. He also earned a name for himself as an artist and painter. Best formerly resided in Lake Murray, South Carolina before moving once again, this time to Hickory, North Carolina. An acting coach, Best taught drama and acting techniques for more than a quarter century in Los Angeles. He also served as artist-in-residence and taught drama at the University of Mississippi (Oxford) for two years prior to his stint on The Dukes of Hazzard. In 2009, he completed his autobiography, Best In Hollywood: The Good, The Bad and The Beautiful. The book, published in 2009 through Bear Manor Media, premiered at the Mid Atlantic Nostalgia Convention in Aberdeen, Maryland.
On November 9, 2014, Best and fellow actor Robert Fuller (along with their wives) attended the 100th birthday celebration of lifelong friend and fellow actor, Norman Lloyd. Best said, "I had the honor to have been directed by Norman in a Hitchcock episode called "The Jar". Having worked with hundreds of directors in my career, I found very few that had Norman's qualities. He was most kind, gracious and patient with his actors. He is in all respects a complete gentleman in his personal life and I found it a genuine pleasure just to be in the presence of such a talented man. I am also doubly honored to consider him my friend. We are so blessed to have such a man among us for so long."
In 1959, Best married his second wife, Jobee Ayers. The couple had two daughters, Janeen and Jojami. They divorced in 1977. Best also had a son, Gary, from a previous marriage. He married his third (and last) wife, Dorothy Collier, in 1986. His niece is actress Jennifer Lyons.
Best died on April 6, 2015, in Hickory, North Carolina from complications of pneumonia. He was 88.Winchester '73 (1950) as Crator
Comanche Territory (1950) as Sam
Kansas Raiders (1950) as Cole Younger
Abbott and Costello Meet the Invisible Man (1951) as Tommy Nelson (Arthur Franz's stand-in)
The Battle at Apache Pass (1952) as Cpl. Hassett
The Beast from 20,000 Fathoms (1953) as an unnamed radar operator
Seminole (1953) as Capt. Gerard
Riders to the Stars (1954) as Sidney K. Fuller
The Caine Mutiny (1954) as Lt. Jorgensen (uncredited)
Forbidden Planet (1956) as an unnamed crewmember
The Naked and the Dead (1958) as Pvt. Rhidges
The Left Handed Gun (1958) as Tom Folliard
Alfred Hitchcock Presents: Death Sentence (1958, Season 3, Episode 30) as Norman Frayne
The Killer Shrews (1959) as Thorne Sherman
Verboten! (1959) as David Brent
Ride Lonesome (1959) as Billy John
The Andy Griffith Show (TV series, 1960–1968) as Jim Lindsey
The Mountain Road (1960) as Niergaard
Bonanza (Episode name-The Fugitive Season 2, Episode 20) as Carl Reagan
The Twilight Zone (The Grave) (1961) (TV series, 1959–1964) as Johhny Rob
The Twilight Zone (The Last Rites of Jeff Myrtlebank) (1962) (TV series, 1959–1964) as Jeff Myrtlebank
The Rifleman (The Day The Town Slept) (1962 Season 4, Episode 139) (TV series, 1958–1963) as Barrett
The Twilight Zone (Jess-Belle) (Feb, 1963) (TV series, 1959–1964) as Billy Ben Turner
Shock Corridor (1963) as Stuart Couter
The Fugitive December 17, 1963 "Terror at High Point"
Combat! (1964) as Trenton
Shenandoah (1965) as Carter
Flipper (1965) as Dr. Peter Kellwin
The Virginian (Letter of The Law (1965)) (TV series, 1962–1971) as Curt Westley
Three on a Couch (1966) as Dr. Ben Mizer
The Guns of Will Sonnett (1967) episode "Meeting at Devil's Fork"
Firecreek (1968) as Drew
Sounder (1972) as Sheriff Young
Ode to Billy Joe (1976) as Dewey Barksdale
Nickelodeon (1976) as Jim
Rolling Thunder (1977) as Texan
Hooper (1978) as Cully
The End (1978) as Pacemaker Patient
Centennial (TV miniseries, 1978) as Hank Garvey
The Dukes of Hazzard (TV series, 1979–1985) as Sheriff Rosco P. Coltrane
The Dukes (TV Cartoon series, 1983) as Sheriff Rosco P. Coltrane
In the Heat of the Night (TV series, 1991) as retired Sheriff Nathadford - Crystal Reel Award, Best Actor
The Dukes of Hazzard: Reunion! (1997 TV movie) as Sheriff Rosco P. Coltrane
The Dukes of Hazzard: Hazzard in Hollywood (2000 TV movie) as Rosco P. Coltrane
Moondance Alexander (2007) as friend and storekeeper of the Alexanders (based on life of real-life daughter Janeen)
Return of the Killer Shrews (2012) as Thorne Sherman
The Sweeter Side of Life (2013) co-starring role as the father of the protagonist