Synaptic communication requires an efficient coupling of vesicle fusion to release neurotransmitter and vesicle retrieval to repopulate the synapse. In synapses of the CNS many proteins involved in exocytosis, endocytosis and refilling of vesicles have been identified. However, little is known about the organization and functioning of synaptic contacts in the enteric nervous system (ENS). We used fluorescent antibodies against presynaptic proteins (synaptobrevin, synaptophysin, synaptotagmin and bassoon) to identify synaptic contacts not only in guinea-pig enteric ganglia but also in the interconnecting fiber strands. Staining patterns were not altered by colchicine (100 muM), ruling out a contribution of protein transport at the time of fixation. Active release sites at fiber intersections and around neuronal cell bodies were labeled with FM1-43 (10 muM) by high K(+) or electric field stimulation (EFS). During a second round of EFS, vesicles were reused, as reflected by dye loss. Destaining rates increased with stimulus frequency (2-30 Hz), reaching a maximum at about 15 Hz, likely caused by synaptic depression at higher frequencies. Tetrodotoxin (TTX, 1 muM) as well as nominally zero external Ca(2+) (2 mM EGTA) prevented all destaining. The readily releasable pool (RRP, a subset of vesicles docked at the membrane and ready to fuse upon [Ca(2+)](i) increase) can be specifically released by a hypertonic challenge (500 mM sucrose). We measured this pool to be approximately 27% of the total recycling pool, remarkably similar to synapses in the CNS. In whole-mount preparations, FM1-43 also reliably labeled active release sites in ganglia, fiber strands and in muscle bundles. The staining pattern indicated that the presynaptic antibodies mainly labeled active sites. The presence of numerous release sites suggests information processing capability within interconnecting fibers. With FM imaging, enteric synaptic function can be monitored independent of any postsynaptic modulation. Although electron microscopy data suggest that ENS synapses may not be as specialized as hippocampal synapses, remarkably similar release properties were measured.