A graduate of Prague's School of Arts and Crafts, in 1936 he created a puppet theater, which was disbanded after the outbreak of WWII. During the war he designed stage sets and illustrated children's books. In 1945 he set up an animation unit with several collaborators at the Prague film studio; they called the unit "Trick Brothers." Trnka specialized in puppet animation, a traditional Czech art form, of which he became the undisputed master. He also created animated cartoons, but it was his puppet animation that made him an internationally recognized artist and the winner of film festival awards at Venice and elsewhere. He wrote the scripts for most of his own films.

Filmography