1. The role of the cytoskeleton in leptin-induced activation of ATP-sensitive K+ (KATP) channels was examined in rat CRI-G1 insulin-secreting cells using patch clamp and fluorescence imaging techniques. 2. In whole cell recordings, dialysis with the actin filament stabiliser phalloidin (10 µM) prevented KATP channel activation by leptin. 3. Application of the actin filament destabilising agents deoxyribonuclease type 1 (DNase 1; 50 µg ml-1) or cytochalasin B (10 µM) to intact cells or inside-out membrane patches also increased KATP channel activity in a phalloidin-dependent manner. 4. The anti-microtubule agents nocodazole (10 microM) and colchicine (100 µM) had no effect on KATP channel activity. 5. Fluorescence staining of the cells with rhodamine-conjugated phalloidin revealed rapid disassembly of actin filaments by cytochalasin B and leptin, the latter action being prevented by the phosphoinositide 3 (PI 3)-kinase inhibitor LY 294002. 6. Activation of KATP channels by the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) was also prevented by phalloidin. This is consistent with the notion that leptin activates KATP channels in these cells by an increase in PtdIns(3,4,5)P3 or a similar 3-phosphorylated phosphoinositol lipid, resulting in actin filament disruption.