Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [35S]GTP gamma S binding in rat brain membranes. Assays were conducted with 0.05 nM [35S]GTP gamma S, incubated with rat cerebellar membranes, 1-30 microM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [35S]GTP gamma S binding increased with increasing concentrations of GDP, with 10-30 microM GDP providing approximately 150-200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [35S]GTP gamma S binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [35S]GTP gamma S binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [35S]GTP gamma S binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [35S]GTP gamma S autoradiography. These results demonstrate that [35S]GTP gamma S binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.