Cell-free fusion between endocytic vesicles has been obtained in a sensitive assay based on the avidin-biotin binding reaction. Chinese hamster ovary cells are allowed to endocytose either avidin-linked beta-galactosidase or biotinylated IgG. Postnuclear supernatant extracts prepared from these cells are incubated at 37 degrees C in the presence of an ATP-regenerating system. Fusion between vesicles from the two extracts permits the avidin-biotin association to occur, so that the amount of avidin-enzyme-biotinylated IgG complex produced is proportional to the amount of fusion between vesicles. The amount of complex formed is measured, after detergent lysis of the vesicles, by an ELISA technique, using a fluorogenic substrate for beta-galactosidase. The fusion process requires ATP hydrolysis and specific cytosolic proteins. The vesicles that fuse appear to be endocytic vesicles populated by the endocytosed proteins within 5 min of their internalization at 37 degrees C. Ionophores and weak bases do not inhibit fusion, suggesting that a pH gradient across the vesicle membrane is not crucial for the fusion process.