Vessel regions with predilection to atherosclerosis have negative wall shear stress due to flow reversal. The flow reversal causes production of superoxides (O₂^-) which scavenge NO, leading to a decrease in NO bioavailability and endothelial dysfunction. Here, we implicate NADPH oxidase as the primary source of O₂^- during full flow reversal. Nitrite production and degree of vasodilation were measured in thirteen porcine common femoral arteries in an ex-vivo system. Nitrite production and vasodilation were determined before and after inhibition of NADPH oxidase, xanthine oxidase or mitochondrial oxidase. NADPH oxidase inhibition with gp91ds-tat or apocynin restored nitrite production and vasodilation during reverse flow. Xanthine oxidase inhibition increased nitrite production at the highest flow rate while mitochondrial oxidase inhibition had no effect. These findings suggest that the NADPH oxidase system can respond to directional changes of flow and is activated to generate O₂^- during reverse flow in a dose-dependent fashion. These findings have important clinical implications for oxidative balance and NO bioavailability in regions of flow reversal in normal and compromised cardiovascular system.