G(i/o)-protein-coupled receptors (GPCRs) ubiquitously inhibit neurotransmission, principally via G??, which acts via a number of possible effectors. GPCR effector specificity has traditionally been attributed to G?, based on G?'s preferential effector targeting in vitro compared with G??'s promiscuous targeting of various effectors. In synapses, however, G?? clearly targets unique effectors in a receptor-dependent way to modulate synaptic transmission. It remains unknown whether G?? specificity in vivo is due to specific G?? isoform-receptor associations or to spatial separation of distinct G?? pathways through macromolecular interactions. We thus sought to determine how G?? signaling pathways within axons remain distinct from one another. In rat hippocampal CA1 axons, GABA(B) receptors (GABA(B)Rs) inhibit presynaptic Ca(2+) entry, and we have now demonstrated that 5-HT(1B) receptors (5-HT(1B)Rs) liberate G?? to interact with SNARE complex C terminals with no effect on Ca(2+) entry. Both GABA(B)Rs and 5-HT(1B)Rs inhibit Ca(2+)-evoked neurotransmitter release, but 5-HT(1B)Rs have no effect on Sr(2+)-evoked release. Sr(2+), unlike Ca(2+), does not cause synaptotagmin to compete with G?? binding to SNARE complexes. 5-HT(1B)Rs also fail to inhibit release following cleavage of the C terminus of the SNARE complex protein SNAP-25 with botulinum A toxin. Thus, GABA(B)Rs and 5-HT(1B)Rs both localize to presynaptic terminals, but target distinct effectors. We demonstrate that disruption of SNARE complexes and vesicle priming with botulinum C toxin eliminates this selectivity, allowing 5-HT(1B)R inhibition of Ca(2+) entry. We conclude that receptor-effector specificity requires a microarchitecture provided by the SNARE complex during vesicle priming.