Phosphoinositides (PI) are important signaling molecules involved in the regulation of vesicular trafficking. We found that phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2)] increase the secretory response triggered by 10 mum Ca(2+) in streptolysin-O-permeabilized insulin-secreting INS-1E cells. In addition, nutrient-induced exocytosis was diminished in intact cells expressing constructs that sequester PI(4,5)P(2) and in cells transfected with constructs that reduce by RNA interference the level of two enzymes involved in PI(4,5)P(2) production, type III PI4-kinase beta and type I phosphatidylinositol 4-bisphosphate 5-kinase-gamma. To clarify the mechanism of action of PI, we investigated the involvement in the regulation of insulin exocytosis of three potential PI targets, phospholipase D1, the Ca(2+)-dependent activator protein for secretion 1, and Munc18-interacting protein 1. Transfection of insulin-secreting cells with plasmids that direct the synthesis of small interfering RNAs capable of reducing the endogenous levels of these proteins inhibited hormone release elicited by glucose- and cAMP-elevating agents without affecting basal release. Our data indicate that the production of PI(4,5)P(2) is necessary for proper control of beta-cell secretion and suggest that at least part of the effect of PI on insulin exocytosis could be exerted through the activation of phospholipase D1, Ca(2+)-dependent activator protein for secretion 1, and Munc18-interacting protein 1.