Background-We have recently reported that hypercholesterolemia reduces aerobic exercise capacity in mice and that this is associated with a reduced endothelium-dependent vasodilator function, endothelium-derived nitric oxide (EDNO) production, and urinary nitrate excretion. These findings led us to test the hypothesis that EDNO production contributes significantly to limb blood flow during exercise and to determine whether loss of EDNO production is responsible for the decline in exercise capacity observed in hypercholesterolemia. Methods and Results: Twelve-week-old wild-type (E+; n=9) and apoE-deficient (E-; n=9) C57BL/6J mice were treadmill-tested to measure indices defining exercise capacity on a metabolic chamber-enclosed treadmill capable of measuring oxygen uptake and carbon dioxide excretion. Urine was collected before and after treadmill exercise for determination of vascular NO production assessed by urinary nitrate excretion. The wild-type mice were then given nitro-L-arginine (E+ LNA) in the drinking water (6 mg/dL) for 4 days before undergoing a second treadmill testing and urinary nitrate measurement. An additional set of 12-week-old wild-type mice was divided into 2 groups: 1 receiving regular water (E+; n=8) and 1 administered LNA for 4 days (E+ LNA; n= 8). These mice, along with an additional set of E- mice (n=8), underwent treadmill testing to determine maximal oxygen uptake (Vo(2)max), The mice were then cannulated such that the tip of the tubing was positioned in the ascending aorta, Fluorescent microspheres (20 000) were infused into the carotid cannula while the mice were sedentary and again while approaching Vo(2)max. When the mice were euthanized, the running muscles were collected and fluorescence intensity was measured to determine the peak-exercise redistribution of blood flow to the running muscles (expressed as percentage of total cardiac output, %COrm) during both states, Both E+ LNA and E- mice demonstrated a markedly reduced postexercise urinary nitrate excretion, aerobic capacity, and %COrm at Vo(2)max compared with E+. Conclusions-EDNO contributes significantly to limb blood flow during exercise. Conditions that reduce EDNO production disturb the hyperemic response to exercise, resulting in a reduced exercise capacity.