Findings in recent years strongly suggest that the stress-inducible gene heme oxygenase (HO)-1 plays an important role in protection against oxidative stress. Although the mechanism(s) by which this protection occurs is poorly understood, we hypothesized that the gaseous molecule carbon monoxide (CO), a major by-product of heme catalysis by HO-1, may provide protection against oxidative stress. We demonstrate here that animals exposed to a low concentration of CO exhibit a marked tolerance to lethal concentrations of hyperoxia in vivo. This increased survival was associated with highly significant attenuation of hyperoxia-induced lung injury as assessed by the volume of pleural effusion, protein accumulation in the airways, and histological analysis. The lungs were completely devoid of lung airway and parenchymal inflammation, fibrin deposition, and pulmonary edema in rats exposed to hyperoxia in the presence of a low concentration of CO. Furthermore, exogenous CO completely protected against hyperoxia-induced lung injury in rats in which endogenous HO enzyme activity was inhibited with tin protoporphyrin, a selective inhibitor of HO. Rats exposed to CO also exhibited a marked attenuation of hyperoxia-induced neutrophil infiltration into the airways and total lung apoptotic index. Taken together, our data demonstrate, for the first time, that CO can be therapeutic against oxidative stress such as hyperoxia and highlight possible mechanism(s) by which CO may mediate these protective effects.