The direct action of somatostatin on smooth muscle was examined in muscle cells isolated from the stomach and intestine of human and guinea pig. Somatostatin inhibited relaxation in gastric but not intestinal muscle cells of the two species, and its mechanism of action was explored in more detail in gastric muscle cells of the guinea pig. Somatostatin inhibited relaxation induced by vasoactive intestinal peptide (VIP, 83 +/- 7%, P less than 0.001) and isoproterenol (85 +/- 5%, P less than 0.001), as well as the concomitant increase in adenosine 3',5'-cyclic monophosphate (cAMP) production [81 +/- 25% inhibition with VIP (P less than 0.02) and 68 +/- 12% inhibition with isoproterenol (P less than 0.01)]. Inhibition of relaxation and cAMP production was abolished by pretreatment of the cells with pertussis toxin. Relaxation induced by the permeant derivative of cAMP, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate, by sodium nitroprusside, which acts by increasing levels of guanosine 3',5'-cyclic monophosphate, or by ATP, which acts by opening of K+ channels, was not affected by somatostatin. The fact that inhibition by somatostatin and its reversal by pertussis toxin was confined to agonists that stimulate an increase in the levels of cAMP implied that somatostatin acts by inhibiting the generation and not the action of cAMP. It is concluded that somatostatin receptors on gastric muscle cells mediate inhibition via a GTP-binding, pertussis-sensitive regulatory protein, Gi, coupled to adenylate cyclase.