Objective: To determine the roles of nitric oxide, endothelin-1 and phosphatidylinositol 3-kinase (PI3-kinase) in acute responses of isolated rat skeletal muscle arterioles to insulin. Methods: Rat cremaster first order arterioles were separated from surrounding tissue, cannulated in a pressure myograph and responses to insulin (4 μU/ml-3.4 mU/ml) were studied without intraluminal blood or flow. Results: Insulin alone did not significantly affect arteriolar diameter. Non-selective antagonism of endothelin receptors, with PD-142893, uncovered insulin-induced vasodilatation (25±8% from baseline at 3.4 mU/ml), which was abolished by inhibition of NO synthesis with NG-nitro-L-arginine (L-NA). Inhibition of NO synthesis alone uncovered insulin-induced vasoconstriction at physiological concentrations (21±5% from baseline diameter at 34 μU/ml), which was abolished by PD-142893. The NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) inhibited insulin-induced vasoconstriction during NOS inhibition, even at a concentration that did not elicit vasodilatation itself. Inhibition of PI3-kinase, an intracellular mediator of insulin-induced NO production, with wortmannin, also uncovered insulin-induced vasoconstriction (13±3% from baseline at 34 μU/ml) that was abolished by PD-142893. Conclusions: Insulin induces both nitric oxide and endothelin-1 activity in rat cremaster first-order arterioles. This study demonstrates for the first time that vasoconstrictive effects of physiological concentrations of insulin during inhibition of NOS activity are mediated by endothelin and that insulin induces endothelin-1-mediated vasoconstriction in isolated skeletal muscle arterioles during inhibition of PI3-kinase. These findings support the hypothesis of altered microvascular reactivity to insulin in conditions of diminished PI3-kinase activity, a prominent feature of insulin resistance.