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While a still scrawny, undersized pre-teen in Morgantown, WV, Don Knotts dreamed of becoming an entertainer, but was too nervous to offer himself as a "single." Purchasing a dummy named Danny, Knotts worked up a ventriloquist act (admittedly stolen from Edgar Bergen) and headed to New York to seek his fortune. After flunking out twice on Major Bowes' Amateur Hour, Knotts returned to Morgantown. He attended West Virginia University as a speech major, intending to become a teacher. He was given a second opportunity to hone his entertaining skills while in Special Services during World War II. He continued pursuing ventriloquism until the fateful night that he threw his dummy into the ocean: "I wanted to get the laughs," Knotts would explain later. And laughs he got as a monologist from both GI and civilian audiences. Never completely conquering his stage fright, Knotts incorporated his nervousness into his act, impersonating such tremulous creatures as a novice TV weatherman and a tongue-tied sportcaster. In New York after the war, Knotts secured work on a local children's show before spending several years on the daytime soap opera Search for Tomorrow.

In 1955, Knotts was cast in two small roles in the Broadway play No Time for Sergeants, which starred another teacher-turned-monologist named Andy Griffith, who would become Knotts' lifelong friend and co-worker. From 1955 through 1960, Knotts was a regular on The Steve Allen Show, provoking uncontrollable bursts of laughter as the bug-eyed, quivering "man on the street." He made his screen debut in the 1958 film version of No Time for Sergeants, re-creating his stage role of the squeaky-voiced coordination therapist. In 1960, he was cast as uptight, self-important, overzealous, magnificently inept deputy Barney Fife on The Andy Griffith Show. This was the role that won Knotts seven Emmies: five during his five-year tenure on the series, and two more when he returned to the show as a guest star in 1966 and 1967. Knotts left the Griffith Show when his contract expired in 1965, hoping to achieve movie stardom. From 1966 through 1971, Knotts ground out a series of inexpensive comedies for Universal (called "regionals" because they played primarily in non-urban and rural theaters). Panned or ignored by the critics on their first release, many of Knotts's starring films, especially The Ghost and Mr. Chicken (1966) and Shakiest Gun in the West (1967), became fan favorites. Arguably, however, the best of Knotts' 1960s films was made at Warner Bros. while he was still an Andy Griffith regular: The Incredible Mr. Limpet, a blend of animation and live-action wherein Knotts was ideally cast as a henpecked husband who metamorphosed into a war-hero fish.

In 1970, Knotts starred in his own TV variety series, which opened to good ratings but ran out of gas after a single season. He resumed his film career, first at Disney, then teamed with Tim Conway in a handful of cheap but amusing B-grade features (The Private Eyes, The Prize Fighter). He also returned to television as self-styled roué Mr. Furley on Three's Company (1979-1984) and as gung-ho principal Bud McPherson on the syndicated What a Country! (1986). That same year, Knotts reprised his most venerable role of Deputy Fife in the made-for-TV movie, Return to Mayberry, the last act of which saw the character becoming the sheriff of Mayberry, NC.

Despite his advancing age, Knotts' output increased in the 1990s and early 2000s. He appeared as a school principal in the Rick Moranis/Tom Arnold comedy Big Bully (1996). Additional roles included a television repairman in Big scribe Gary Ross's 1998 directorial debut, Pleasantville; the voice of T.W. Turtle in Cats Don't Dance, the voice of Turkey Lurkey in the 2005 Disney comedy Chicken Little, and a turn as "The Landlord" on an episode of That '70s Show that represented a deliberate throwback to Three's Company. Knotts spent much of his final decade teaming up with his old friend and co-star, Tim Conway, on the voiceovers for the Hermie and Friends series, contemporary Christian animated videos about a bunch of colorful insects.

The world lost Don Knotts on February 25, 2006; he died in Beverly Hills, CA. In his final years, Knotts's appearances on the big or the small screen were greeted with the sort of appreciative laughter and applause that is afforded only to a genuine television icon.