Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein ?? dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. G?? binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of G?? on SNAP-25, peptides based on the sequence of SNAP-25 were screened for G?? binding. Peptides that bound G?? were subjected to alanine scanning mutagenesis to determine their relevance to the G??-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of G?? with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to G????. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for G??, but it still interacted with synaptotagmin-1 in a Ca²? -dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.