Phosphatidylinositol 3-kinases (PI3Ks) have a central role in pancreatic -cell function. Downstream events include the regulation of K_ATP channel activity, insulin secretion, gene transcription and cell survival. Less data are available on the 3-phosphorylated inositol lipids (3-PI's) that are the primary products of these kinases. We characterized these PI3K-products in insulin secreting HIT-T15 cells and were able to demonstrate, for the first time, the presence of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂). We then showed that glucose can significantly increase PtdIns(3,4,5)P₃, PtdIns(3,4)P₂ and notably PtdIns(3,5)P₂. We investigated the mechanism(s) whereby these molecules are generated under both basal and glucose-stimulated conditions. We postulated that insulin exocytosis could drive the rises in 3-PI's. In our experimental system we could detect a rise in insulin secretion within 1 minute of glucose stimulation, thus allowing the possibility that early rises in 3-PIs are regulated by secreted insulin. This was confirmed as blockade of the -cell insulin receptor completely abrogated the glucose-mediated increase of all three lipids, driving their concentrations below basal levels. Using primary pancreatic islets and either blockade of the insulin receptor or antibodies to insulin we verified that basal insulin secretion is responsible for the maintenance of 3-PIs. Therefore, autocrine insulin signaling, a feature compromised in diabetes, is essential to upregulate both basal and glucose-stimulated levels of a vital family of second messengers that preserve and drive pancreatic -cell function.