In order to differentiate the effects of hyperoxia and barotrauma in the pathogenesis of acute neonatal lung injury, piglets were either hyperventilated (Paco2, 15-20 torr) for 48 hours with 100% oxygen (Group I), hyperventilated with 21% oxygen (Group II), normally ventilated (Paco2, 40-45 torr) with 100% oxygen (Group III), or normally ventilated with 21% O2 (Group IV) and compared to unventilated controls. Pulmonary function was tested, and biochemical indicators of lung injury were analyzed in tracheo-bronchial aspirates at 0, 24, and 48 hours. Bronchoalveolar lavage fluid was analyzed for surfactant composition and activity at the end of the study. At 48 hours, hyperoxic, hyperventilated piglets had significantly decreased dynamic lung compliance (30%) and increased pulmonary resistance (16%), aspirate cell count (190%), elastase activity (88%), albumin (214%), and total protein (150%) concentration. Qualitative light microscopy showed moderate to severe atelectasis, fibrinous exudate, edema, and inflammation. Normoxic, hyperventilated animals had comparable changes in pulmonary mechanics, but significantly milder cellular, biochemical, and morphologic changes. In hyperoxic, normocarbic animals pulmonary physiologic, cellular, and biochemical variables changed comparably to hyperoxic, hyperventilated animals; the pathologic changes were intermediate between hyperoxic, hyperventilated and normoxic, hyperventilated piglets. Normoxic, normocarbic animals had no significant changes in most variables over 48 hours; on morphologic examination their lungs were similar to unventilated controls and showed only mild edema. Surfactant had normal biophysical activity in all animals. Our results demonstrate that hyperoxia causes more significant physiologic, inflammatory, and histologic changes than barotrauma alone. Future attempts to prevent lung injury in neonates should be directed primarily at oxygen toxicity.