Wortmannin (5–100 nm), a specific phosphatidyinositol 3-kinase inhibitor, augmented 8 mm glucose-induced insulin secretion from control Sprague Dawley rat islets in a dose-dependent manner. This effect persisted after its removal from the perifusion medium; however, this augmenting effect was reduced by the calcium channel inhibitor nitrendipine or by lowering the glucose level to 3 mm. Wortmannin amplified insulin release induced by the combination of 6–8 mm glucose plus 1 μm carbachol; however, it had no effect on phorbol ester- orα -ketoisocaproate-induced insulin secretion. The potentiating action of wortmannin on 8 mm glucose-induced release was duplicated by LY294002. Wortmannin had no effect on glucose usage rates or inositol phosphate accumulation in[ ³H]inositol-prelabeled islets. Of particular significance, although 50 nm wortmannin potentiated 8 mm glucose-induced secretion from islets of lean Zucker control rats, the fungal metabolite had little effect on 8 mm glucose-induced release from islets of insulin-resistant Zucker fatty rats. These findings support the concept that the same biochemical process, inhibition of phosphatidyinositol 3-kinase, that causes peripheral tissue insulin resistance enhances β-cell sensitivity to glucose and produces a compensatory increase in insulin secretion from these cells. The efficacy of wortmannin depends on the in vivo status of the donor’s insulin signaling pathways. This elegant biochemical control mechanism in β-cells ensures the maintenance of glucose homeostasis despite a reduction in insulin action on peripheral tissues.