The therapeutic usefulness of O2 is limited primarily by pulmonary O2 toxicity, and there are no effective therapeutic modalities other than avoidance for prevention of this toxicity. In the present study, superoxide dismutase, an enzyme that catalyzes the dismutation of superoxide anion to less toxic forms, as well as reduced glutathione and succinate, two agents that protect against central nervous system O2 toxicity, were evaluated for ability to protect against the development of pulmonary O2 toxicity in the rat. Pulmonary O2 toxicity was evaluated by measuring lung clearance of serotonin in the isolated, perfused lung. Depression of serotonin clearance has recently been shown to be an index of early lung O2 poisoning. Rats received 60 nmole of superoxide dismutase per kg of body weight, 16 mmole of reduced glutathione per kg, 12 mmole of succinate per kg, or an equal volume of normal saline in a single intraperitoneal injection 45 min before a 60-min exposure to either 100 per cent O2 at 4 atm absolute or air. Lungs were then isolated, ventilated, and perfused using a recirculating system for measurement of serotonin clearance. Gross lung morphologic features, pulmonary arterial and ventilation pressures, and ratio of lung dry weight to wet weight were similar in all groups exposed to air and to O2. The mean ±SE fractional clearance by the lungs of rats exposed to air was 0.79 ± 0.04 and did not vary with pretreatment. After exposure to 100 per cent O2 at 4 atm absolute, there was a 29 per cent decrease (0.56 ± 0.05; P < 0.001) in serotonin clearance. Pretreatment with reduced glutathione or succinate was not protective. In contrast, serotonin clearance by the lungs of rats exposed to 100 per cent O2 and treated with superoxide dismutase was 0.70 ± 0.04 (P < 0.02 versus O2 plus normal saline, reduced glutathione, or succinate; P > 0.10 versus exposure to air). These results suggest that pretreatment of rats with superoxide dismutase may protect against the development of pulmonary O2 toxicity and that one mechanism of lung damage by hyperoxia may be increased biologic production of the superoxide anion.