Instillation of intratracheal surfactant is known to limit the morbidity and mortality of patients and animals with oxidant-induced lung injury. In this study we quantified the antioxidant properties of natural lung surfactant (NLS), consisting of 90% lipid and 10% protein, and of calf lung surfactant extract (CLSE) consisting of 99% lipid and 1% protein. NLS, but not CLSE, contained significant amounts of superoxide dismutase (SOD) and catalase activities (7 U SOD/ μmol phospholipid (PL) and 1 U catalase/μmol PL). More than 90% of the SOD activity was abolished by 1 mM KCN, suggesting that this was the CuZn form of the enzyme. In addition, NLS significantly reduced extracellular H2O2 without losing its ability to reach minimum surface tensions below 1 dyn/cm upon dynamic compression. The NLS scavenging of H2O2 could not be accounted for by albumin. The presence of catalase and SOD activities in NLS was also verified by activity stains of proteins separated by native polyacrylamide gel electrophoresis. Intratracheal installation of 7 ml of NLS (308 μmol PL) into rabbits significantly increased SOD content in type II cells isolated 12 h later. It is concluded that, in addition to promoting alveolar stability, installation of pulmonary surfactant may offer significant protection to the alveolar epithelium by scavenging extracellularly generated partially reduced oxygen species and by enhancing intracellular antioxidant enzyme content. © 1990.