Heme oxygenase (HO)-1, which catalyzes heme breakdown, is induced by oxidative stress and may protect against oxidative injury. We hypothesized that induction of HO-1 by hemoglobin (Hb) in the lung would protect the rat from pulmonary O2 toxicity. Rats given intratracheal (i.t.) Hb showed lung-specific induction of HO-1 by 8 h by Western analysis. Rats were then pretreated for 8 h before 60 h of exposure to 100% O2 with either IT normal saline, HB, or Hb plus the HO-1 inhibitor tinprotoporphyrin (SnPP). Both the Hb + O2 and Hb + O2 + SnPP animals had less lung injury than normal saline controls as indicated by lower pleural fluid volumes and wet-to-dry weight ratios (P < 0.01). The improvement in injury in the two Hb-treated groups was the same despite a 61% decrease in HO enzyme activity in the Hb + SnPP group after 60 h of O2. In addition, inhibition of HO activity with SnPP alone before O2 exposure did not augment the extent of hyperoxic lung injury. These results demonstrate that IT Hb induces lung HO-1 in the rat and protects against hyperoxia; however, the protection is not mediated by increased HO enzyme activity.