1. Opioid (mu, delta, kappa) and cannabinoid (CB1, CB2) receptors are coupled mainly to Gi/Go GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir, or utilize different pools, of G proteins. 2. The stimulation of [35S]GTPgammaS binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of mu- and delta-opioid receptors led to stimulation of [35S]GTPgammaS binding by either mu-selective (DAMGO) or delta-selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes. 3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either mu- or delta-opioid receptors. 4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and delta-opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress mu- and delta-opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptor subtype. 5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.