Presynaptic inhibition mediated by G protein-coupled receptors may involve a direct interaction between G proteins and the vesicle fusion machinery. The molecular target of this pathway is unknown. We demonstrate that Gbetagamma-mediated presynaptic inhibition in lamprey central synapses occurs downstream from voltage-gated Ca(2+) channels. Using presynaptic microinjections of botulinum toxins (BoNTs) during paired recordings, we find that cleavage of synaptobrevin in unprimed vesicles leads to an eventual exhaustion of synaptic transmission but does not prevent Gbetagamma-mediated inhibition. In contrast, cleavage of the C-terminal nine amino acids of the 25 kDa synaptosome-associated protein (SNAP-25) by BoNT A prevents Gbetagamma-mediated inhibition. Moreover, a peptide containing the region of SNAP-25 cleaved by BoNT A blocks the Gbetagamma inhibitory effect. Finally, removal of the last nine amino acids of the C-terminus of SNAP-25 weakens Gbetagamma interactions with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. Thus, the C terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, may represent a target of Gbetagamma for presynaptic inhibition.