G-proteins are important mediators of hormonal inhibition of insulin secretion. To characterize the pertussis toxin–sensitive substrates present in HIT cell membranes, we performed immunoblots with specific antisera and found evidence for the presence of GIα1, GIα2, GIα3, and three forms of GO. We observed that pertussis toxin–sensitive substrates mediate all of the effects of SRIF, and a major portion of the effects of EPI, on insulin secretion from rat islets during static incubations. These results agree with our previously reported studies examining phasic glucose-induced insulin secretion from HIT cells. To ascertain whether inhibition of adenylate cyclase, presumably involving coupling of the catalytic subunit to GI, may be a common mechanism for both hormones, we studied the effects of 8-bromo-cyclic AMP and found that this agent partially prevented the inhibitory effects of both hormones. We also observed that the inhibitory effects of SRIF and EPI on insulin were nonadditive, that both hormones were additive to nickel chloride during inhibition of insulin release, and that they noncompetitively inhibited glipizide-induced insulin secretion through pertussis toxin-sensitive mechanisms. Together, these results suggest that both hormones exert their effects on insulin secretion at multiple G- protein -regulated sites including adenylate cyclase and sites distal to the glipizide-binding site on the KATP channel.