Tetanus toxin and clostridial neurotoxins type B, D, F, and G inhibit intracellular Ca(2+)-dependent neurotransmitter release via the specific proteolytic cleavage of vesicle-associated membrane protein (VAMP)/synaptobrevin, a highly conserved 19-kDa integral protein of the small synaptic vesicle membrane. This results in the release of the larger part of the cytosolic domain of this synaptic protein into the cytoplasm. Microinjection of synthetic peptides corresponding to this fragment into identified presynaptic neurons of Aplysia californica led to a potent, long lasting, and dose-dependent inhibition (approximately 50% at 10 MicroM) of acetylcholine release, probably by hindering endogenous VAMP/synaptobrevin from interacting with synaptic proteins involved in exocytosis. Structure activity studies showed that this effect is confined to the N-terminal domain of VAMP/synaptobrevin isoform II and is related to the presence of a proline-rich motif (PGGPXGX3PP or PAAPXGX3PP). At higher concentrations, the inhibitory effect was lower and only transient, suggesting that the N-terminal proline-rich domain of VAMP/synaptobrevin plays opposing roles in neurotransmitter release very likely by interacting with different synaptic proteins. This probably occurs by disruption of the recently reported in vitro VAMP-synaptophysin interaction that involves the N-terminal domain of VAMP II and was proposed to hinder synatophysin-related formation of a fusion pore. The observed recovery of neurotransmitter release following injection of high concentration of N-terminal fragments of VAMP II brings a strong in vivo support to this hypothesis. The minimum active peptide GPGGPQGGMQPPREQS could be used for rationally designing potent synthetic blockers of neurotransmission.