Synaptic vesicles are released from membranes during incubation at 37 degrees C in the presence of ATP (adenosine triphosphate). The donor membranes are a rapidly sedimenting fraction derived from the neuroendocrine cell line PC12 (pheochromocytoma 12). These starting membranes contain the synaptic vesicle proteins, synaptophysin and SV2, and the endosomal markers transferrin receptor and cation-independent MPR (mannose 6-phosphate receptor). Incubating the membranes in vitro increased the amount of organelles that migrate as synaptic vesicles in velocity sedimentation gradients. The synaptic vesicle fractions that contain both synaptophysin and SV2 do not contain endosomal markers. A synaptic vesicle increase in vitro is time-, cytosol-, ATP- and temperature-dependent and is inhibited by NEM (N-ethylmaleimide), BFA (brefeldin A) and aluminum fluoride, but not GTP gamma S (guanosine-5'O-C3-thiotriphosphate). The production of synaptic vesicles under these conditions is unlike the de novo generation of vesicles from endosomes (1). Incubation in vitro under the conditions described here may allow the final stages of synaptic vesicle formation, uncoating or undocking, to occur but not the initiation of formation de novo.