CB1 receptors mediate the CNS effects of Δ9-tetrahydrocannabinol and synthetic cannabinoids. Previous studies have investigated cannabinoid-mediated G-protein activity in a subset of brain regions thought to mediate the behavioral effects of cannabinoids, but a detailed regional comparison of the effects of multiple ligands has not been conducted. This study used a novel approach, Statistical Parametric Mapping (SPM), to analyze 3D reconstructed brain images derived from agonist-stimulated [35S]GTPγS autoradiography in a whole-brain unbiased manner. SPM analysis demonstrated regional differences in the relative efficacies of cannabinoid agonists methanandamide (M-AEA), CP55,940 (CP) and WIN55,212-2 (WIN) in CB1+/+ mouse brains. To assess the potential contribution of novel cannabinoid binding sites, experiments were performed in CB1−/− mouse brains. SPM analysis revealed that the aminoalkylindole WIN, but not the bicyclic cannabinoid CP or the endocannabinoid analogue M-AEA, stimulated [35S]GTPγS binding in cortex, hippocampus, hypothalamus, amygdala, cerebellum and certain brainstem areas (dorsal tegmental complex and locus coeruleus). No differences between WIN-stimulated G-protein activity and basal activity were found in basal ganglia. Pharmacological experiments using the CB1 antagonist SR141716A in CB1+/+ mice showed that SR141716A blocked WIN-stimulated G-protein activity in all brain regions, suggesting that it binds to both CB1 and putative non-CB1 sites. These studies show ligand and region-specific cannabinoid-mediated G-protein activity at both CB1 and non-CB1 sites and demonstrate that SPM is a powerful approach for the analysis of reconstructed brain imaging data derived from agonist-stimulated [35S]GTPγS autoradiography.