The signal transduction mechanisms involved in insulin secretion by the beta-cell are poorly understood. Glucose, the main physiological secretagogue, needs to be metabolized, but the identity of the intracellular messengers which couple glucose metabolism and insulin exocytosis is controversial. We now report the identification of phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3), the end-product of phosphatidylinositol 3-kinase phosphorylation of polyphosphoinositides, in islets and in an insulin-secreting clonal beta-cell line, RINm5F, using a combination of thin layer chromatography and high performance liquid chromatography analyses and by demonstrating that sequential deacylation and deglyceration of PtdIns(3,4,5)P3 yields inositol 1,3,4,5-tetrakisphosphate. Unlike other cell types, significant levels of PtdIns(3,4,5)P3 were detected in beta-cells under non-stimulatory conditions. Insulin secretagogues (28 mM glucose + 0.5 mM carbachol) caused a rapid and transient increase in PtdIns(3,4,5)P3 levels which peaked at 2-5 min, corresponding to peak early phase insulin release.