This study investigates the effects of the islet hormones, insulin (INS), glucagon (GLU) and somatostatin (SOM) on acetylcholine (ACh)-evoked amylase secretion and calcium (Ca2+) mobilization in the isolated rat pancreas. Stimulation of pancreatic segments and acini with either INS, GLU or SOM resulted in small increases of amylase output compared to much large increases in enzyme output with ACh. Combinations of the peptide hormones with ACh resulted in enhanced secretory responses compared to the effects obtained with either ACh or each of the islet hormone alone. Genistein, the tyrosine kinase inhibitor, evoked a decrease in amylase output from pancreatic segments. It had no effect on the ACh evoke secretory response but it markedly inhibited the potentiation of the islet hormones with ACh. In pancreatic acinar cells either INS, GLU or SOM elicited moderate increases in amylase output compared to much larger responses with ACh. Furthermore, the islet hormones failed to potentiate the secretory effect of ACh in pancreatic acini. In fura-2 AM loaded acinar cells both INS and GLU evoked small increases in intracellular free calcium concentration [Ca2+]i compared to a much larger elevation with ACh. Both INS and GLU enhanced the ACh-evoked [Ca2+]i. Genistein elicited a decrease in [Ca2+]i both in the absence and presence of both INS and GLU. It also decreased the rise in [Ca2+]i resulting from the combined presence of ACh with both INS and GLU. SOM had no significant effect on the ACh-induced [Ca2+]i. When genistein was combined with ACh and SOM there was a decrease in [Ca2+]i compared to the response obtained with SOM and ACh alone. The results indicate that both tyrosine kinase and cellular Ca2+ seem to be the intracellular mediators associated with the enhanced secretory responses obtained with a combination of the islet hormones with ACh. Finally, our results using immunohistochemical techniques confirm the presence of INS-, GLU- SOM- and ACh-immunoreactive cells in the endocrine and neural elements of the rat pancreas.