Epidermal growth factor (EGF) has been shown to accelerate fetal lung maturation in rabbits, lambs, and rhesus monkeys in vivo and increase surfactant synthesis in vitro. Its effect on the maturation of the lung antioxidant enzyme system, however, is unknown. We studied the effect of EGF (10 nM) on 19-d fetal rat lung explant cultures in serum-free medium in air/5% CO2 or >90% O2/5% CO2 compared with similarly grown control cultures in air or hyperoxia at 72 h. Fetal lung activities of super-oxide dismutase and catalasc were unchanged by EGF in air, whereas glutathione peroxidase activity was significantly decreased (p < 0.05 versus air control). However, in hyperoxia, EGF-treated fetal lung cultures had significantly elevated superoxide dismutase and catalase activities (p < 0.01) versus O2-exposed controls, and glutathione peroxidase activity similar to that of controls. The mRNA levels for all the antioxidant enzymes showed patterns similar to the enzyme activities except in the case of Cu, Zn-superoxide dismutase mRNA, which increased in EGF-air cultures. EGF decreased the rate of 3H-choline incorporation into disaturated phosphatidylcholinc in air (p < 0.01 versus air control), but increased disaturated phosphatidylcholine synthesis in response to hyperoxia (p < 0.01 versus O2 control). The histologic appearance of EGF-treated cultures in O2 was superior to that of O2-exposed controls, which showed thickened scptal walls, decreased surfactant in the air spaces, and epithelial cell mitochondrial swelling. EGF therefore accelerates antioxidant enzyme and disaturated phosphatidylcholine maturation under hyperoxic conditions and protects fetal rat lung cultures from hyperoxic injury. This accelerated O2-dependent maturation by EGF occurs at the pretranslational level. These findings could have clinical implications for premature infants requiring O2 therapy and at risk for bronchopulmonary dysplasia because of immature pulmonary antioxidant defenses.