Nitric oxide (NO) synthase inhibition reduces leg glucose uptake during cycling without reducing leg blood flow (LBF) in young, healthy individuals. This study sought to determine the role of NO in glucose uptake during exercise in individuals with type 2 diabetes. Nine men with type 2 diabetes and nine control subjects matched for age, sex, peak pulmonary oxygen uptake (VO(2) peak), and weight completed two 25-min bouts of cycling exercise at 60 +/- 2% VO(2) peak, separated by 90 min. N(G)-monomethyl-L-arginine (L-NMMA) (total dose 6 mg/kg) or placebo was administered into the femoral artery for the final 15 min of exercise in a counterbalanced, blinded, crossover design. LBF was measured by thermodilution in the femoral vein, and leg glucose uptake was calculated as the product of LBF and femoral arteriovenous glucose difference. During exercise with placebo, glucose uptake was not different between control subjects and individuals with diabetes; however, LBF was lower and arterial plasma glucose and insulin levels were higher in individuals with diabetes. L-NMMA had no effect on LBF or arterial plasma glucose and insulin concentrations during exercise in both groups. L-NMMA significantly reduced leg glucose uptake in both groups, with a significantly greater reduction (P = 0.04) in the diabetic group (75 +/- 13%, 5 min after L-NMMA) compared with the control group (34 +/- 14%, 5 min after L-NMMA). These data suggest a greater reliance on NO for glucose uptake during exercise in individuals with type 2 diabetes compared with control subjects.