To determine the functional role of nitric oxide (NO) in regulating vascular conductance to skeletal muscle, female Wistar rats (277 ± 4 g; n = 7) were run at a speed established to yield VO2max (i.e., treadmill; 60 m/min, 10% grade). Regional vascular conductances (VC, ml/min/100g/mmHg) were measured using radiolabeled microspheres during exercise before and after NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg i.a.). NOS inhibition increased (p < 0.05) mean arterial pressure from the pre-exercise baseline value of 137 ± 2 to 168 ± 5 mmHg and attenuated (p < 0.05) the vasodilator response to acetylcholine injections (10 mg/kg i.a.), demonstrating reduced NOS function. During exercise, NOS inhibition reduced VC to the kidneys and splanchnic organs. In addition, VC was reduced in 20 of the 28 individual hindlimb muscles or muscle parts (average −25 ± 2%, P < 0.05). Also, these reductions in VC were correlated linearly with the percentage of slow-twitch oxidative (SO) and fast-twitch oxidative glycolytic (FOG) fiber types found in each muscle [ΔVC = −0.0082(%SO + %FOG) - 0.0105, r = 0.66, P < 0.001], similar to that found previously for moderate intensity exercise (20 m/min, 10% grade). The reduction in VC for the total hindlimb musculature produced by NOS inhibition during high intensity exercise (ΔVC = −0.37 ± 0.06) was significantly greater (p < 0.05) than that found at rest (ΔVC = −0.15 ± 0.03) and during moderate intensity exercise (ΔVC = −0.28 ± 0.04 ml/min/100g/mmHg). These results suggest that during high intensity exercise NO contributes substantially to the regulation of VC within and among the muscles of the rat hindlimb with its relative contribution being greater in muscles that have a high oxidative capacity. Moreover, L-NAME reduces VC in an exercise intensity dependent manner. Supported in part by NIH-HL-50306, HL-57226, AG-11535