Regulators of G-protein signaling (RGS)-insensitive (RGSi) G-protein alpha subunits can be used to indirectly determine the function of endogenous RGS proteins in native cells. This article describes the application of RGSi Galpha subunits to the study of endogenous RGS function in central nervous system (CNS) neurons. Presynaptic inhibition of neurotransmitter release was reconstituted in primary neurons using RGSi Galpha(i/o) subunits, whereas postsynaptic regulation of potassium channels was reconstituted using RGSi chimeras of Galpha(q) and Galpha(i). These studies have shown that endogenous RGS proteins are essential for the rapid termination of some G-protein-mediated signals in CNS neurons, whereas these proteins are much less important for the regulation of other signals. Together, these techniques have helped reveal the complexity of RGS regulation of CNS function.