Heterotrimeric G-proteins mediate many receptor-coupled signal transduction processes and the cellular concentrations of G-proteins are modulated by several factors, including development, activity, and drugs. The mechanisms causing changes in G-protein concentrations are mostly unknown. The purpose of this study was to determine if G-proteins could be proteolyzed by calpain, a calcium-activated neutral protease that has been linked with neuronal plasticity. In membranes prepared from rat cerebral cortex, calpain rapidly cleaved the α-subunit of Go but did not hydrolyze ß-subunits. Comparisons of the proteolysis of different α-subunits revealed that they were differentially susceptible to calpain-induced proteolysis in the order ofαs > αo > αq > αi. Preincubation of cortical membranes with GTPγS, which binds to Gα and causes its dissociation from the ßγ dimer, reduced calpain-mediated proteolysis of αo. Lithium, the primary treatment for mania, enhanced the calpain-mediated proteolysis of αo in the heterotrimeric state but did not affect proteolysis of dissociated, GTPγS-bound αo. These results demonstrate that proteolysis by calpain is a potential mechanism by which cellular G-protein concentrations can be regulated.