During exocytosis, certain phospholipids may act as regulators of secretion. Here, we used several independent approaches to perturb the phosphatidylinositol-4,5-bisphosphate[PI(4,5) P-2] level in bovine chromaffin cells to investigatehowchanges of plasmalemmal PI(4,5) P-2 affect secretion. Membrane levels of PI(4,5) P-2 were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5) P-2. The specific PI(4,5) P-2 signal was enriched in submicrometer-sized clusters. In parallel patch-clamp experiments on intact cells, we measured the secretion of catecholamines. Overexpression of phosphatidylinositol-4-phosphate-5-kinase Igamma, or infusion of PI(4,5) P-2 through the patch pipette, increased the PI(4,5) P-2 level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5) P-2 from the membrane and abolished secretion. An inhibitor of phosphatidylinositol-3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, led to a transient increase in the PI(4,5) P-2 level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5) P-2 decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5) P-2 levels led to correlated changes in the size of two releasable vesicle pools, whereas their fusion kinetics remained unaffected. We conclude that during both short- and long-term manipulations of PI(4,5) P-2 level secretion scales with plasma membrane PI(4,5) P-2 content and that PI(4,5) P-2 has an early effect on secretion by regulating the number of vesicles ready for release.