Background and purpose: We have recently shown that the phytocannabinoid Delta(9)-tetrahydrocannabivarin (Delta(9)-THCV) and the CB1 receptor antagonist AM251 increase inhibitory neurotransmission in mouse cerebellum and also exhibit anticonvulsant activity in a rat piriform cortical (PC) model of epilepsy. Possible mechanisms underlying cannabinoid actions in the CNS include CB1 receptor antagonism (by displacing endocannabinergic tone) or inverse agonism at constitutively active CB1 receptors. Here, we investigate the mode of cannabinoid action in [S-35]GTP gamma S binding assays. Experimental approach: Effects of Delta(9)-THCV and AM251 were tested either alone or against WIN55,212-2-induced increases in [S-35]GTP gamma S binding in mouse cerebellar and PC membranes. Effects on non-CB receptor expressing CHO-D-2 cell membranes were also investigated. Key results: Delta(9)-THCV and AM251 both acted as potent antagonists of WIN55,212-2-induced increases in [S-35]GTP gamma S binding in cerebellar and PC membranes (Delta(9)-THCV: pA(2) = 7.62 and 7.44 respectively; AM251: pA(2) = 9.93 and 9.88 respectively). At micromolar concentrations, Delta(9)-THCV or AM251 alone caused significant decreases in [S-35]GTP gamma S binding; Delta(9)-THCV caused larger decreases than AM251. When applied alone in CHO-D-2 membranes, Delta(9)-THCV and AM251 also caused concentration-related decreases in G protein activity. Conclusions and implications: Delta(9)-THCV and AM251 act as CB1 receptors antagonists in the cerebellum and PC, with AM251 being more potent than Delta(9)-THCV in both brain regions. Individually, Delta(9)-THCV or AM251 exhibited similar potency at CB1 receptors in the cerebellum and the PC. At micromolar concentrations, Delta(9)-THCV and AM251 caused a non-CB receptor-mediated depression of basal [S-35]GTP gamma S binding.