Prolonged exposure to hyperoxia represents a serious danger to cells, yet little is known about the specific cellular factors that affect hyperoxia stress. By screening the yeast deletion library, we have identified genes that protect against high-O2 damage. Out of approx. 4800 mutants, 84 were identified as hyperoxia-sensitive, representing genes with diverse cellular functions, including transcription and translation, vacuole function, NADPH production, and superoxide detoxification. Superoxide plays a significant role, since the majority of hyperoxia-sensitive mutants displayed cross-sensitivity to superoxide-generating agents, and mutants with compromised SOD (superoxide dismutase) activity were particularly vulnerable to hyperoxia. By comparison, factors known to guard against H2O2 toxicity were poorly represented amongst hyperoxia-sensitive mutants. Although many cellular components are potential targets, our studies indicate that mitochondrial glutathione is particularly vulnerable to hyperoxia damage. During hyperoxia stress, mitochondrial glutathione is more susceptible to oxidation than cytosolic glutathione. Furthermore, two factors that help maintain mitochondrial GSH in the reduced form, namely the NADH kinase Pos5p and the mitochondrial glutathione reductase (Glr1p), are critical for hyperoxia resistance, whereas their cytosolic counterparts are not. Our findings are consistent with a model in which hyperoxia toxicity is manifested by superoxide-related damage and changes in the mitochondrial redox state.