Calf-brain coated vesicles were incubated with ATP and a cytosol fraction. As much as 90% of the clathrin was selectively released within 10 min at 37 °C without detectable proteolysis. This uncoating process required the presence of both ATP and cytosol. Empty cages of clathrin could also be dissociated in a similar manner. A nonhydrolyzable analogue, 5'-adenylylimidodiphosphate (AMPPNP), would not substitute for ATP. Clathrin wasdissociated from coats in a form unable to reassemble into cages under standard conditions. These reactions may reflect a segment of a clathrin-coated vesicle cycle in which coats are removed from vesicles after budding. © 1982, Rockefeller University Press., All rights reserved.