Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca²⁺ responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 and wortmannin, as well as genetic deletion of the PI3K gamma isoform (PI3K), regulates Ca²⁺ responses and the Ca²⁺-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3K involvement in Ca²⁺ responses induced in these cells by cholecystokinin-8 (CCK) and carbachol. The PI3K inhibitors inhibited both Ca²⁺ influx and mobilization from intracellular stores induced by stimulation of acini with physiologic and pathologic concentrations of CCK, as well as with carbachol. PI3K inhibition facilitated the decay of [Ca²⁺]i oscillations observed in individual acinar cells. The PI3K inhibitors decreased neither CCK-induced IP₃ production nor IP₃-induced Ca²⁺ mobilization suggesting that the effect of PI3K inhibition is not through IP₃ or IP₃ receptors. PI3K inhibition did not affect Ca²⁺ mobilization induced by thapsigargin, a specific inhibitor of sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacologic and genetic PI3K inhibition on [Ca²⁺]i signals, suggesting SERCA as a downstream target of PI3K. Pharmacologic PI3K inhibition as well as genetic deletion of PI3K both increased the amount of Ca²⁺ in intracellular stores during CCK stimulation. Finally, addition of PI3K product, phosphatidylinositol 3,4,5-trisphosphate, to permeabilized acini significantly attenuated Ca²⁺ reloading into the ER. The results indicate that PI3K regulates Ca²⁺ signaling in pancreatic acinar cells through its inhibitory effect on SERCA.