ATP-sensitive potassium channels (K(ATP)) are involved in a diverse array of physiologic functions including protection of tissue against ischemic insult, regulation of vascular tone, and modulation of insulin secretion. To improve our understanding of the role of K(ATP) in these processes, we used a gene-targeting strategy to generate mice with a disruption in the muscle-specific K(ATP) regulatory subunit, SUR2. Insertional mutagenesis of the Sur2 locus generated homozygous null (Sur2(-/-)) mice and heterozygote (Sur2(+/-)) mice that are viable and phenotypically similar to their wild-type littermates to 6 weeks of age despite, respectively, half or no SUR2 mRNA expression or channel activity in skeletal muscle or heart. Sur2(-/-) animals had lower fasting and fed serum glucose, exhibited improved glucose tolerance during a glucose tolerance test, and demonstrated a more rapid and severe hypoglycemia after administration of insulin. Enhanced glucose use was also observed during in vivo hyperinsulinemic euglycemic clamp studies during which Sur2(-/-) mice required a greater glucose infusion rate to maintain a target blood glucose level. Enhanced insulin action was intrinsic to the skeletal muscle, as in vitro insulin-stimulated glucose transport was 1.5-fold greater in Sur2(-/-) muscle than in wild type. Thus, membrane excitability and K(ATP) activity, to our knowledge, seem to be new components of the insulin-stimulated glucose uptake mechanism, suggesting possible future therapeutic approaches for individuals suffering from diabetes mellitus.