Jesse Donald Knotts was born in Morgantown, West Virginia, the youngest of four sons born to farmer William Jesse Knotts and his wife, Elsie Luzetta Knotts (nee Moore). His parents were married in Spraggs, Pennsylvania. His English paternal ancestors immigrated to America in the 17th century, originally settling in Queen Anne's County, Maryland. Knotts' brothers were named Willis, William, and Ralph.

In 1986, Don Knotts reunited with Andy Griffith in the made-for-television film Return to Mayberry, again reprising his Barney Fife role. In early 1987, Knotts joined the cast of the first-run syndication comedy What a Country!, playing Principal Bud McPherson for the series' remaining 13 episodes. The sitcom was produced by Martin Rips and Joseph Staretski, who had previously worked on Three's Company. In 1988, Knotts joined Andy Griffith in another show, playing the recurring role of pesky neighbor Les Calhoun on Matlock until 1992.  After that, Knotts's roles were sporadic, including a cameo appearance in the film Big Bully (1996) as the principal of the high school. In 1998, Knotts played a small but pivotal role as a mysterious TV repairman in Pleasantville. That year, his home town of Morgantown, West Virginia, changed the name of the street formerly known as South University Avenue (U.S. Route 119) to Don Knotts Boulevard on "Don Knotts Day." Also that day, in honor of Knotts's role as Barney Fife, he was named an honorary deputy sheriff with the Monongalia County Sheriff's Department.  Knotts was recognized in 2000 with a star on the Hollywood Walk of Fame. He continued to act on stage, but much of his film and television work after 2000 was as voice talent. In Ghost World (2001) Enid (Thora Birch) is asked why she painted a portrait of Don Knotts. She replies, "I just like Don Knotts." The portrait in the film may be the last visual image of Knotts in a motion picture. In 2002, he appeared again with Scooby-Doo in the video game Scooby-Doo! Night of 100 Frights. (Knotts also spoofed his appearances on that show in various promotions for Cartoon Network and in a parody on Robot Chicken, where he was teamed with Phyllis Diller). In 2003, Knotts teamed up with Tim Conway again to provide voices for the direct-to-video children's series Hermie and Friends, which continued until his death. In 2005, he was the voice of Mayor Turkey Lurkey in Chicken Little (2005), his first Disney movie since 1979. On September 12, 2003, Knotts was in Kansas City in a stage version of On Golden Pond when he received a call from John Ritter's family telling him that his former Three's Company co-star had died of an aortic dissection that day. Knotts and his co-stars attended the funeral four days later. Knotts had appeared with Ritter one final time in a cameo on 8 Simple Rules for Dating My Teenage Daughter. It was an episode that paid homage to their earlier television series. Knotts was the last Three's Company star to work with Ritter.  During this period of time, macular degeneration in both eyes caused the otherwise robust Knotts to become virtually blind. His live appearances on television were few. In 2005, Knotts parodied his Ralph Furley character while playing a Paul Young variation in a Desperate Housewives sketch on The 3rd Annual TV Land Awards. He parodied that part one final time, "Stone Cold Crazy", an episode of the sitcom That '70s Show. In the show, Knotts played Fez and Jackie's new landlord. This was his last live-action television appearance. His final role was in Air Buddies (a 2006 direct-to-video sequel to Air Bud), voicing the sheriff's deputy dog, Sniffer.

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