Nitric oxide (NO) appears to play a role in contraction-stimulated glucose uptake in isolated rodent skeletal muscle; however, no studies have examined this question in humans. Seven healthy men completed two 30-min bouts of supine cycling exercise at 60 +/- 2% peak pulmonary oxygen uptake (VO2 peak), separated by 90 min of rest. The NO synthase inhibitor N(G)-monomethyl-L-arginine ([L-NMMA]; total dose 5 mg/kg body weight) or saline (control) were administered via the femoral artery for the final 20 min of exercise in a randomized blinded crossover design. L-Arginine (5 mg/kg body weight) was co-infused during the final 5 min of each exercise bout. Leg blood flow (LBF) was measured by thermodilution in the femoral vein, and leg glucose uptake was calculated as the product of LBF and femoral arteriovenous (AV) glucose difference. L-NMMA infusion significantly (P < 0.05) reduced leg glucose uptake compared with control (48 +/- 12% lower at 15 min, mean +/- SE). The reduction in glucose uptake was due solely to a decrease in AV glucose difference, as there was no effect of L-NMMA infusion on LBF during exercise. Co-infusion of L-arginine restored glucose uptake during L-NMMA infusion to levels similar to control. These results indicate that NO production contributes substantially to exercise-mediated skeletal muscle glucose uptake in humans independent of skeletal muscle blood flow.