The functional role of nitric oxide (NO) release in regulating blood flow (BF) to exercising skeletal muscle was studied in conscious male Sprague-Dawley rats (603 +/- 28 g; n = 6). In this study, BF was measured using radiolabeled microspheres during treadmill exercise (10% grade, 20 m/min) before and after NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (30 mg/kg ia). After NOS inhibition, mean arterial blood pressure increased from resting baseline values and the duration of vasodilator responses to acetylcholine (ACh) injections (3.0 and 10.0 micrograms/kg ia) was diminished (P < 0.05), demonstrating reduced NOS function. During exercise, BF to the kidneys and organs of the gut was reduced after NOS inhibition. In addition, BF was reduced in 16 of the 28 individual hindquarter muscles or muscle parts. Moreover these reductions in BF were linearly correlated with the estimated sum of the percentage of fast-twitch oxidative glycolytic (FOG) and slow-twitch oxidative (SO) types of fibers found in each muscle [delta BF = -1.1 (%SO + %FOG) + 16.4; r = 0.88, P < 0.001]. These results suggest that NO-mediated vasodilation contributes to the BF responses within and among the muscles of the rat's hindquarters during exercise.