In rat pancreatic acinar cells epidermal growth factor (EGF) and insulin increase both basal and cholecystokinin (CCK-OP) stimulated amylase release in vitro (1) as a long term function of this tissue. Here we show that preincubation of isolated plasma membranes with EGF or with insulin leads to increased incorporation of the GTP-photoaffinity analogue [alpha-32P]GTP-gamma-azidoanilide into 40/41 kDa proteins and to reduction of pertussis toxin- (PT) catalyzed [alpha-32P]ADP-ribosylation of three 40/41 kDa proteins which had been previously identified as Gi1, Gi2 and Gi3 (2). In the presence of GTP gamma S, EGF- and insulin-induced inhibition of PT-mediated [alpha-32P]ADP-ribosylation of 40/41 kDa proteins is eliminated. EGF enhances cholera toxin- (CT) mediated ADP-ribosylation of all three 40/41 kDa Gi-proteins as well as of five 45 and four 48/50 kDa proteins, which had been previously identified as Gs-proteins (2), whereas insulin has no effect. We conclude from our data that both EGF and insulin interact with the same Gi-proteins as CCK-OP does, whereas EGF additionally interacts with nine Gs-proteins. It is likely that one, two or all three 40/41 kDa Gi-proteins are involved in insulin- and EGF-induced potentiation of CCK-OP-stimulated enzyme secretion. In addition interaction of EGF with Gs-protein could play a role in the potentiation of CCK-OP-induced enzyme secretion from pancreatic acinar cells.