Objectives. We sought to determine the importance of nitric oxide (NO) production in maintaining coronary blood flow during exercise in hearts with collateral-dependent myocardium. Background. Coronary collateral vessels demonstrate endothelium-mediated NO-dependent vasodilation in response to agonists such as acetylcholine. However, the contribution of endogenous NO production to maintaining vasodilation of coronary collateral vessels during exercise has not been previously studied. Methods. Collateral vessel growth was induced in 13 chronically instrumented dogs by intermittent 2-min occlusions, followed by permanent occlusion of the left anterior descending coronary artery (LAD). One week after permanent LAD occlusion, myocardial blood flow was measured with microspheres during rest and treadmill exercise at 6.4 km/h at a 15% grade. Measurements were then repeated after blockade of NO production with N-nitro-L-arginine (LNNA) (20 mg/kg body weight intravenously). Results. LNNA caused a 62 ± 4% (mean ± SEM) inhibition of the coronary vasodilation produced by acetylcholine. During rest conditions, LNNA caused a slight decrease in blood flow to the collateral region (p = NS), with no change in normal zone blood flow. During exercise, LNNA caused a decrease in mean blood flow to the collateral region (from 2.24 ± 0.19 to 1.78 ± 0.26 ml/min per g after LNNA, p < 0.05). This decrease resulted from a near doubling of the collateral vascular resistance (p < 0.05), with a trend toward an increase in small vessel resistance in the collateral zone. LNNA also reduced myocardial blood flow to the normal region during exercise (from 2.99 ± 0.24 to 2.45 ± 0.28 ml/min per g, p < 0.05) as the result of a 44 ± 13% increase in coronary vascular resistance (p < 0.05). Conclusions. NO contributes to the maintenance of coronary collateral blood flow during exercise. In contrast to the normal heart, endogenous NO production also maintains blood flow in remote myocardial regions during exercise. These results suggest that control of blood flow during exercise in normal myocardium is altered by the presence of an occluded coronary artery.