In his first American film, Give Me Your Heart (1936), released in Great Britain as Sweet Aloes, Knowles was cast as a titled Englishman of means.  While making The Charge of the Light Brigade (1936) at Lone Pine, California, he befriended Errol Flynn, whom he closely resembled and whose acquaintance he had made in England when both were under contract to Warner Bros. at Teddington Studios. Since that film, in which Knowles played the part of Capt. Perry Vickers, the brother of Flynn’s Maj. Geoffrey Vickers, he was cast more frequently as straitlaced characters alongside Flynn’s flamboyant ones, notably as Will Scarlet in The Adventures of Robin Hood (1938). Both actors starred as well in Four’s a Crowd, also in 1938. More than two decades after Flynn’s death, biographer Charles Higham accused Flynn of having been a fascist sympathizer and Nazi spy. Knowles, who had served in World War II as a flying instructor in the RCAF, came to Flynn’s defense, writing Rebuttal for a Friend as an epilogue to Tony Thomas’ Errol Flynn: The Spy Who Never Was (Citadel Press, 1990) ISBN 0-8065-1180-X.

He was a freelance film actor from 1939 until his last film appearance in 1973. In the 1940s, he was known for playing protagonists in a number of horror films, including The Wolf Man (1941) and Frankenstein Meets the Wolfman (1943). He was also cast as comic foils in a number of comedies such as Abbott and Costello’s Who Done It? (1942) and Hit the Ice (1943), and as the leading man in romantic comedies like Lady in a Jam (1942), in which he co-starred with Irene Dunne. His voice also appears uncredited in the Three Stooges film Punch Drunks (1934), as an announcer heard on a radio. He also appeared opposite Jack Kelly in a 1957 episode of the television series Maverick called “The Wrecker”, which was based on a Robert Louis Stevenson adventure and also featured James Garner. He also appeared in Have Gun – Will Travel in 1960 as Phileas Fogg in the episode “Foggbound.” Arguably one of his best film roles was also one of his last: his genial portrayal of the rancher John Tunstall in the John Wayne Western Chisum (1970).  Knowles was inducted into the Hollywood Walk of Fame.  Knowles died at West Hills Hospital in West Hills, California on 23 December 1995.