In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. The part played by Ca2+ released from intracellular stores in the regulation of secretion is well established; however, the role of Ca2+ influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (≥10 μM) elicited an additional component of exocytosis despite reducing Ca2+ influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (∼70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 μM) was potentiated (∼45%) by wortmannin. Exocytosis stimulated by 1 μM acetylcholine in the absence of extracellular Ca2+ was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca2+ entry through store-operated Ca2+ channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd3+ (2 μM), but not by inhibition of noncapacitative Ca2+ entry by SB203580 (10 μM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca2+ influx.