We studied the interaction between somatostatin receptors and inhibitory GTP binding protein in rat cerebrocortical membranes. Guanine nucleotides reduced [¹²⁵I-Tyr¹] somato-statin binding to cerebrocortical membranes in a dose-dependent manner with rank order of potency being guanyl-5′-yl-imidodiphosphate (Gpp (NH) p) >GTP>GMP. Maximum reduction of the binding to 32% of control was observed in the presence of 10^-5M Gpp (NH) p. Scatchard analysis of the labeled somatostatin binding revealed that the decrease in the binding by Gpp (NH) p was due to the decrease in the binding affinity for somatostatin. Divalent cations, such as Mg⁺⁺, Mn⁺⁺ and Ca⁺⁺, caused an increase in labeled somatostatin binding to membranes with the maximum binding observed at a concentration of 10, 10, 1 mM, respectively. However, Na⁺ decreased a labeled somatostatin binding in a dose-dependent manner, and half maximum inhibition of the binding was observed at 10mM Na⁺. Moreover, Gpp (NH) p and Na⁺ lowered labeled somatostatin binding in an additive fashion. When cerebrocortical membranes were treated at 37°C for 40min with various concentrations of Islet-Activating-Protein (IAP), which had been preactivated with dithiothreitol, sub-sequent labeled somatostatin binding to the membranes was decreased in a dose-dependent manner. 30μ/ml TAP treatment caused a decrease in the binding to 50% of control, which was characterized by the decreased binding affinity without a significant change in the binding capacity. Furthermore, exposure of IAP plus NAD to cerebrocortical membranes caused ADP-ribosylation of a membrane protein with Mr=41,000 on autoradiogram. Such an TAP treatment of cerebrocortical membranes abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. These results suggest that somatostatin receptors in the brain couple to inhibitory GTP binding protein, which mediates adenylate cyclase inhibition by somatostatin.