Abstract—The mechanism of oxygen-induced cerebral vasoconstriction has been sought for more than a century. Using genetically altered mice to enhance or disrupt extracellular superoxide dismutase (EC-SOD, SOD3), we tested the hypothesis that this enzyme plays a critical role in the physiological response to oxygen in the brain by regulating nitric oxide (NO·) availability. Cerebral blood flow responses in these genetically altered mice to changes in Po2demonstrate that SOD3 regulates equilibrium between superoxide (·O2−) and NO·, thereby controlling vascular tone and reactivity in the brain. That SOD3 opposes inactivation of NO·is shown by absence of vasoconstriction in response to Po2in the hyperbaric range in SOD3+/+mice, whereas NO-dependent relaxation is attenuated in SOD3−/−mutants. Thus, EC-SOD promotes NO·vasodilation by scavenging ·O2−while hyperoxia opposes NO·and promotes constriction by enhancing endogenous ·O2−generation and decreasing basal vasodilator effects of NO·.