Systemic hyperinsulinemia induces vasodilation in human skeletal muscle. This vasodilation contributes to insulin-stimulated glucose uptake and has been found to be reduced in various insulin-resistant states. The mechanism of the effect of insulin on vascular tone is not completely understood. We hypothesized that activation of the sodium-potassium pump (Na+, K(+)-ATPase) located in endothelial or smooth muscle cells would be involved in the insulin-mediated vasodilation. Therefore, in 24 healthy, nonsmoking, nonobese, normotensive volunteers, we infused ouabain, a specific inhibitor of Na+, K(+)-ATPase, into the brachial artery before and during euglycemic hyperinsulinemia. As expected, insulin (systemic concentrations, approximately 700 [low] and 1400 [high] pmol.L-1) induced vasodilation in the control arm (forearm blood flow [FBF, plethysmography] from 1.6 +/- 0.2 to 2.1 +/- 0.4 mL.dL-1.min-1 [low insulin] and from 1.6 +/- 0.2 to 2.1 +/- 0.2 [high insulin], P < .05 for both), but the increase in FBF was abolished in the ouabain-infused forearm (from 1.3 +/- 0.1 to 1.4 +/- 0.2 mL.dL-1.min-1 [low] and from 1.3 +/- 0.1 to 1.3 +/- 0.1 [high], P = NS). Ouabain-induced increases in forearm potassium release were partly reversed by insulin. To investigate whether the mechanism of action could be at the endothelial level, we infused NG-monomethyl-I-arginine (L-NMMA), an inhibitor of endothelial nitric oxide synthase (0.05, 0.1, and 0.2 mg.dL-1.min-1) intra-arterially in 12 subjects and induced a clear dose-dependent decrease of FBF from 1.7 +/- 0.2 to 1.2 +/- 0.1 mL.dL-1.min-1 (P < .01). In contrast, after ouabain (and continued insulin) infusion, L-NMMA had no effect on FBF (from 1.6 +/- 0.4 to 1.5 +/- 0.3 mL.dL-1.min-1, n = 6, P = .66). These results demonstrate that insulin induces vasodilation by stimulation of Na+, K(+)-ATPase. This activation of Na+, K(+)-ATPase could occur at the level of the endothelium rather than that of vascular smooth muscle and contributes to the endothelium-dependent vasodilator response to insulin.