The temperature-sensitive mutant of Drosophila, shibire(ts-1), which is normal at 19 degrees C, but in which endocytosis is reversibly blocked at 29 degrees C, was used to deplete synapses of vesicles by inducing transmitter release while membrane retrieval was blocked. When the synapse was kept at 29 degrees C for 8 min, complete vesicle depletion occurred. However, no compensatory increase in the terminal plasma membrane, either as invaginations or evaginations, was observed. Also, no internalized membranous compartment, such as cisternae or coated vesicles, appeared. No invaginations or out-pocketings were seen along the axon between release sites, and no evidence for elongation of the whole axon was found. Thus, the vesicle membrane compartment became unobservable as a result of transmitter release. Depleted synapses were observed by electron microscopy at various times after lowering the temperature, so that the process of synaptic vesicle reformation could be observed. In the first 2–3 min at 19 degrees C, gradually enlarging uncoated invaginations of the plasma membrane were observed. Between 5– 10 min at 19 degrees C, these invaginations pinched off to form large cisternae. Newly formed synaptic vesicles were observed associated with these cisternae by an electron-dense material. Between 10–20 min at 19 degrees C, the number of synaptic vesicles increased, while the size of the cisternae decreased. Within 30 min, the full complement of vesicles had reappeared. No involvement of the coated vesicle pathway in synaptic vesicle reformation was observed. The data suggest that synaptic vesicle membrane is dissembled at the time of transmitter release and then is reassembled at sites along the plasma membrane and internalized in the form of large cisternae, from which new vesicles are formed.