Cannabinoid compounds, including the major psychoactive component of marihuana, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), have been widely established as being inhibitory on a broad array of humoral and cell-mediated immune responses. The presence of cannabinoid receptors has been identified recently on mouse spleen cells, which possess structural and functional characteristics similar to those of the G-protein coupled cannabinoid receptor originally identified in rat brain. These findings, together with those demonstrating that Delta(9)-THC inhibits adenylate cyclase in splenocytes, strongly suggest that certain aspects of immune inhibition by cannabinoids may be mediated through a cannabinoid receptor-associated mechanism. The objective of the present studies was to determine whether inhibition of adenylate cyclase is relevant to mouse spleen cell immune function and, if so, whether this inhibition is mediated through a G(i)-protein coupled mechanism as previously described in neuronal tissue. Spleen cell activation by the phorbol ester phorbol-12-myristate-13-acetate (PMA), plus the calcium ionophore ionomycin, produced a rapid but transient increase in cytosolic cAMP, which was inhibited completely by immunosuppressive concentrations of Delta(9)-THC (22 mu M) and the synthetic bicyclic cannabinoid CP-55940 (5.2 mu M), which produced no effect an cell viability. Inhibition by cannabinoids of lymphocyte proliferative responses to PMA plus ionomycin and the sheep erythrocyte (sRBC) IgM antibody-forming cell (AFC) response, was abrogated completely by low concentrations of dibutyryl-cAMP (10-100 mu M). Inhibition of the sRBC AFC response by both Delta(9)-THC (22 mu M) and CP-55940 (5.2 mu M) was also abrogated by preincubation of splenocytes for 24 hr with pertussis toxin (0.1-100 ng/mL). Pertussis toxin pretreatment of spleen cells was also found to directly abrogate cannabinoid inhibition of adenylate cyclase, as measured by forskolin-stimulated accumulation of intracellular cAMP. These results indicate that inhibition of the sRBC AFC response by cannabinoids is mediated, at least in part, by inhibition of adenylate cyclase through a pertussis toxin-sensitive G(i)-protein coupled cannabinoid receptor. Additionally, these studies further support the premise that cAMP is an important mediator of lymphocyte activation.