The nuclear gene for manganese-containing superoxide dismutase (MnSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) of yeast mitochondria was mapped on chromosome VIII and inactivated by gene disruption. The resulting mutant lacked any protein cross-reacting with anti-MnSOD antibodies, and its mitochondria exhibited less than 1% of the cyanide-insensitive superoxide dismutase activity found in mitochondria of the wild-type parent strain. In the absence of oxygen, the mutant grew as rapidly as the wild-type parent. However, increasing concentrations of oxygen led to a progressive inhibition of growth. The properties of this mutant provide direct evidence that MnSOD contributes to the natural protection of cells against oxygen toxicity.