Presynaptic receptors that are coupled to heterotrimeric G-proteins are found throughout the brain and are responsible for modulating synaptic transmission. At least 10 G-protein-coupled receptors (GPCRs) reduce transmission in hippocampal neurons. Additionally, hippocampal neurons express up to 17 different Galpha, Gbeta, and Ggamma subunits, making for a striking array of possible heterotrimer compositions and GPCR-heterotrimer interactions. The identity of the Galpha subunit is likely a critical determinant in coupling specificity between GPCRs and their molecular effectors mediating presynaptic inhibition. We studied the role of four Galpha(i/o) subunits (Galpha(o1), Galpha(i1,) Galpha(i2), and Galpha(i3)) in mediating presynaptic inhibition in hippocampal neurons by expressing pertussis toxin-insensitive (PTx-ins) Galpha(i/o) mutants. PTx treatment of these cells disrupts coupling of endogenous subunits, leaving only the mutant Galpha subunits to couple with native GPCRs and betagamma subunits. Successful rescue of presynaptic inhibition indicates that the expressed mutant Galpha subunit can couple to the GPCR of interest. All four PTx-ins Galpha subunits rescued presynaptic inhibition by adenosine A1 receptors. A PTx-ins Galpha subunit also rescued adenosine A1-mediated inhibition of spontaneous vesicle fusion frequency. Of the remaining GPCRs tested, cannabinoid CB1, somatostatin, and GABA(B) receptors displayed an alpha subunit-dependent selectivity in binding to G-protein heterotrimers, whereas group III metabotropic glutamate receptor-mediated inhibition was not rescued by expression of any of the four PTx-ins Galpha subunits. Differential coupling of G-protein alpha subunits may be a means of achieving specificity between different GPCRs and their molecular targets for mediating presynaptic inhibition.