We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.