Born in Toledo, Ohio in 1928 to Greek immigrant parents, Markopoulos began making 8 mm films at an early age.
He attended USC Film School in the late 1940s, and went on to become a co-founder — with Jonas Mekas, Shirley Clarke, Stan Brakhage and others — of the New American Cinema movement.
In 1967, he and his partner Robert Beavers left the United States for permanent residence in Europe.
Once ensconced in self-imposed exile, Markopoulos withdrew his films from circulation, refused any interviews, and insisted that a chapter about him be removed from the second edition of Visionary Film, P. Adams Sitney's seminal study of American avant-garde cinema.
Markopoulos dedicated his film (A)lter (A)ction to Rosa von Praunheim.