H eme oxygenase (HO) activity leads to accumulation of the antioxidant bilirubin, and degradation of the prooxidant heme. Moderate overexpression of the inducible form, HO-1, is associated with protection against oxidative injury. However, the role of HO-2 in oxidative stress has not been explored. We evaluated survival, indices of oxidative injury, and lung and HO expression in HO-2 null mutant mice exposed to > 95% O2 compared with wild-type controls. Similar basal levels of major lung antioxidants were observed, except that the knockouts had a twofold increase in total glutathione content. Despite increased HO-1 expression from HO-1 induction, knockout animals were sensitized to hyperoxia-induced oxidative injury and mortality, and also had significantly increased markers of oxidative injury before hyperoxic exposure. Furthermore, during hyperoxia, lung hemoproteins and iron content were significantly increased without increased ferritin, suggesting accumulation of available redox-active iron. These results demonstrate that the absence of HO-2 is associated with induction of HO-1 and increased oxygen toxicity in vivo, apparently due to accumulation of lung iron. These results suggest that HO-2 functions to augment the turnover of lung iron during oxidative stress, and that this function does not appear to be compensated for by induction of HO-1 in the knockouts.