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Born and raised in a wealthy family with turbulent changes caused by his father's lifestyle, Macnee was raised by his mother, Dorothea Mary, and her lesbian partner.
Graduating from Eton College (despite a near-expulsion due to his involvement in a gambling ring), Macnee began his career as an extra in the 1938 film, Pygmalion (starring the film's star and director, Leslie Howard). World War II interrupted his acting career as he served in the Royal Navy, after which Macnee entered into a drama school for additional training in Britain.
Macnee landed additional minor and extra roles in some of the first televised movies in the latter 1940s, including Wuthering Heights, Macbeth, Othello and Scrooge. Seeking more substantial roles, Macnee left to see work in Canada and the neighboring United States.
Macnee became more successful from his experience on the North American continent, and returned for a time to Britain to produce a television series on the noted figure Winston Churchill.
Returning to America (later becoming an American citizen in 1959) for roles in what were to become classic 1950s television programs such as The Twilight Zone, Playhouse 90 and Rawhide, Macnee landed the leading role that would define him as an icon with the coming of the "secret agent" spy genre: the character of John Steed, the derby-wearing, umbrella-carrying secret agent in the British TV series The Avengers. Steed became the focus of the show after the first season, playing opposite a series of female partners such as Honor Blackman, Diana Rigg, and Linda Thorson. Macnee lent his costume designing skills to the series as well. The series ran from 1961 to 1969, and Macnee later reprised the character of Steed in The New Avengers, which ran from 1976 to 1977.
During and after the immediate success of The Avengers, Macnee has successfully used his talents and his Avengers status in minor and supporting roles in many American feature films and television shows of the 1970s, 1980s, and 1990s, including Matt Helm (a very loose adaptation of the spy character), Night Gallery, The Hardy Boys/Nancy Drew Mysteries, Hotel and Frasier.
Macnee portrayed the mysterious Iblis in the Original Series two-part episode "War of the Gods," and also lent his voice talents to the opening narration of the series, which began with "Life here...began out there." Macnee would also co-star in other Glen A. Larson series, including the short-lived NightMan and Larson's most successful series to-date, Magnum P.I..
Macnee's voice talents also featured in a 1998 film revival of The Avengers, not as John Steed, but in a cameo role as "Invisible Jones".
Macnee has been married to Baba Majos de Nagyzsenye, his third wife, since 1988. He has two children, Rupert and Jenny, by his first marriage to Barbara Douglas (from 1942 to 1956). His second wife (1965-1969) was actress Kate Woodville. The notable British television magician David Nixon was his cousin.