Previous studies have indicated that cannabinoids inhibit presynaptic neurotransmitter release in brain through CB1 receptors. To examine this issue in a primary neuronal culture system, rat cerebellar granule cells (CGCs) were prepared. [35S]GTPgammaS binding assays in saponin-permeabilized CGCs showed that G-protein activation by the CB1 agonist, WIN55212-2, and adenosine A1 agonist, phenylisopropyladenosine, was maximal during the second week in culture. Delta9-tetrahydrocannabinol stimulated [35S]GTPgammaS binding to a lesser degree than WIN55212-2, and the antagonists SR141716A and AM281 acted as inverse agonists in intact CGCs, but not in CGC membrane preparations. Ten micromolar WIN55212-2 and Delta9-tetrahydrocannabinol decreased depolarization-evoked efflux of [3H]-D-aspartate from CGCs by 32% and 13%, respectively. SR141716A and AM281 increased [3H]-D-aspartate release by 28%. The fatty acid amide hydrolase (FAAH) inhibitor phenylmethylsulfonyl fluoride (PMSF) and the anandamide uptake inhibitor AM404 inhibited transmitter release, implying that the antagonist effects were mediated by blockade of endocannabinoid activity. Levels of endocannabinoids (both anandamide and 2-arachidonyl glycerol [2-AG]) in extracts of the cells and cell incubation buffer were increased by PMSF pre-treatment. Depolarization with KCl significantly decreased the amount of anandamide and 2-AG in PMSF-treated CGCs. These results suggest that endogenous cannabinoids inhibit neurotransmitter release in CGCs, which may also release endocannabioids upon neural stimulation.