Previously, we have demonstrated that somatostatin mediates all of its inhibitory effects on glucose-induced insulin secretion from the HIT-T15 cell through pertussis toxin-sensitive G-proteins and that the membrane fraction of this clonal line of pancreatic β-cells contains six such proteins: G(iα1), G(iα2), G(iα3), and three forms of G(oα). To determine the specificity of somatostatin receptor-G-protein coupling in HIT-T15 cells, we examined the ability of antisera specific for the COOH-terminus of G(α) subtypes to inhibit somatostatin-induced augmentation of membrane GTPase activity. GTPase activity increased in membranes as a function of GTP. At all concentrations of GTP studied, 1 μmol/l somatostatin stimulated GTPase activity. Pertussis-toxin pretreatment prevented the effects of somatostatin. Antisera selective for G(oα) subtypes reduced the effects of somatostatin on GTPase activity (GTPase activity in absence of antisera, 125 ± 3% of control; in the presence of antisera 976, 110 ± 2% of control; n = 13, P < 0.001), whereas antisera directed against G(iα1), G(iα2), G(iα3), and G(sα) were without effect. Somatostatin also significantly prevented cyclic AMP accumulation during perifusion with 11.1 mmol/l glucose through a pertussis toxin-sensitive mechanism. These data indicate that the somatostatin receptor couples to G(oα) in the HIT-T15 cell and suggest that G(oα) may link somatostatin to cyclic AMP metabolism in pancreatic β- cells.