The modulation of ATP-sensitive K+ channel (K(ATP)) by insulin plays a role in neuromuscular disorders associated to altered K+ homeostasis. However, the mechanisms by which insulin modulates K(ATP) channels are not known. Here, the insulin-dependent 3Na+/2K+ ATP-ase and Pi-3 kinase pathways were explored by using patch-clamp techniques. High and low affinity inhibition of K(ATP) channels by ouabain was observed in the insulin-stimulated and resting fibers, respectively. The 9A5 antibody directed against the alpha1-subunit of the pump inhibited the K(ATP) channel in the resting fibers but fails to inhibit it in the insulin-stimulated fibers. In contrast, the RT2NKATPabr, an alpha2-subunit specific antibody, inhibited the K(ATP) channels in the insulin-stimulated fibers failing to inhibit it in the resting fibers. The insulin-dependent stimulation of K(ATP) channel was prevented by Pi-3 kinase inhibitors Wortmannin and LY294002. In conclusion, insulin stimulating the 3Na+/2K+ ATP-ase activates K(ATP) channels through a membrane-delimited interaction thus controlling the K+ homeostasis. The Pi-3 kinase is the intracellular insulin signal linking the glucose homeostasis to the K(ATP) channel.