The effects of insulin and acetylcholine (ACh) on amylase secretion, transmembrane movement of 45Ca2+ and K+, membrane potential and cyclic nucleotide levels in the isolated mouse pancreas were investigated. Insulin alone had no effect on either amylase secretion or 45Ca2+ fractional efflux but it markedly potentiated the ACh-evoked amylase secretion and significantly reduced the ACh-induced 45Ca2+ fractional efflux. These effects were dose related. Insulin evoked a small membrane hyperpolarization and an increase in K+ efflux. The islet hormone had virtually no effect on ACh-induced membrane depolarization but it markedly enhanced the ACh-elicited K+ efflux. Both insulin and ACh had marked time-dependent effects on the metabolism of adenosine 3',5'-cyclic monophosphate (cyclic AMP). Insulin increased and ACh decreased cyclic AMP concentration when applied separately. However, when added together, insulin and ACh caused a rapid and sustained elevation of cyclic AMP levels. Superfusion of mouse pancreatic fragments with an exogenous lipid-soluble derivative of cyclic AMP (dibutyryl adenosine 3',5'-cyclic monophosphate) caused dose-dependent increases in amylase secretion. Dibutyryl cyclic AMP also markedly enhanced, in a dose-dependent manner, the ACh-evoked amylase secretion. It is concluded that insulin may exert its potentiating action on ACh-evoked amylase output in the mouse pancreatic acinar cells by elevating both cytoplasmic Ca2+ and cyclic AMP levels.