The control of vascular tone during exercise is highly complex and integrated. Specifically, in regards to the contribution of nitric oxide (NO), the observed magnitude and muscle fiber-type dependency of the NO contribution to exercise hyperemia may differ depending on the timing of NO synthase (NOS) inhibition with respect to the exercise bout (i.e., administration prior to vs. during exercise). We tested the hypothesis that, in the presence of prior cyclooxygenase inhibition (indomethacin, 5 mg/kg(-1)), NOS inhibition (N(G)-nitro-L-arginine methyl ester, L-NAME; 10 mg/kg) administered during submaximal treadmill exercise would blunt blood flow and vascular conductance (VC) in the hindlimb muscle(s) of the rat with the greatest reductions in blood flow and VC occurring in the predominantly oxidative muscles. Adult female Wistar rats (n = 10, age: 3-4 mo) ran on a motor-driven treadmill (20 m/min, 10% grade). Total and regional hindlimb muscle blood flow and VC were determined via radiolabeled microspheres before (control) and after L-NAME administration during exercise. L-NAME reduced (P < 0.05) total hindlimb muscle blood flow (control: 123 + or - 10, L-NAME: 103 + or - 7 ml x min(-1) x 100g(-1)) and VC (control: 0.95 + or - 0.09, L-NAME: 0.63 + or - 0.05 ml x min(-1) x 100g(-1) x mmHg(-1)). There was a significant correlation (r = 0.51, P < 0.05) between the absolute reductions in VC after L-NAME and the percent sum of type I and IIa fibers in the individual muscles and muscle parts; however, there was no correlation (P = 0.62) when expressed as blood flow. Surprisingly, the highly oxidative muscles demonstrated a marked ability to maintain oxygen delivery, which differs substantially from previous reports of L-NAME infusion prior to exercise in these muscles. The demonstration that NO is an important regulator of blood flow and VC in the rat hindlimb during treadmill exercise, but that the fiber-type dependency of NO is altered markedly when NOS inhibition is performed during, vs. prior to, exercise, lends important insights into the integrated nature of vascular control during exercise.