The mechanism by which leptin increases ATP-sensitive K+ (KATP) channel activity was investigated using the insulin-secreting cell line, CRI-G1. Wortmannin and LY 294002, inhibitors of phosphoinositide 3-kinase (PI3-kinase), prevented activation of KATP channels by leptin. The inositol phospholipids phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate (PtdIns(3,4,5)P3) mimicked the effect of leptin by increasing KATP channel activity in whole-cell and inside-out current recordings. LY 294002 prevented phosphatidylinositol bisphosphate, but not PtdIns(3,4,5)P3, from increasing KATP channel activity, consistent with the latter lipid acting as a membrane-associated messenger linking leptin receptor activation and KATP channels. Signaling cascades, activated downstream from PI 3-kinase, utilizing PtdIns(3,4,5)P3 as a second messenger and commonly associated with insulin and cytokine action (MAPK, p70 ribosomal protein-S6 kinase, stress-activated protein kinase 2, p38 MAPK, and protein kinase B), do not appear to be involved in leptin-mediated activation of KATP channels in this cell line. Although PtdIns(3,4,5)P3 appears a plausible and attractive candidate for the messenger that couples KATP channels to leptin receptor activation, direct measurement of PtdIns(3,4,5)P3 demonstrated that insulin, but not leptin, increased global cellular levels of PtdIns(3,4,5)P3. Possible mechanisms to explain the involvement of PI 3-kinases in KATP channel regulation are discussed.