To test the hypothesis that nitric oxide production is essential for endogenous vascular remodeling in ischemic skeletal muscle, 22 New Zealand white rabbits were chronically instrumented with transit-time flow probes on the common iliac arteries and underwent femoral ligation to produce unilateral hindlimb ischemia. Iliac blood flow and arterial pressure were recorded at rest and during a graded exercise test. An osmotic pump (Alzet, 2ML2) connected to a femoral arterial catheter continuously delivered L-NAME (nitric oxide synthase inhibitor) or a control solution (D-NAME or phenylephrine) to the ischemic limb over a 2 week period. One, 3 and 6 weeks after femoral ligation, maximal treadmill exercise blood flow in the ischemic limb was reduced compared to baseline in each group. However, the L-NAME group had a significantly (p<0.05) lower maximal exercise blood flow compared to the control group for the duration of the study (week 6: L-NAME 48±4, Control 60±5 ml/min). Consistent with the reduction in maximal blood flow response, the duration of voluntary exercise was also substantially (p<0.05) shorter in the group that received L-NAME (539±67 vs 889±87 sec). Resting blood flow was unaffected by femoral ligation in either group. The results of this study show that endogenous vascular remodeling, which partially alleviated the initial deficit in blood flow, was interrupted by nitric oxide synthase inhibition. Therefore, we conclude that nitric oxide is essential for endogenous collateral development and angiogenesis in ischemic skeletal muscle in the rabbit.