Total glutathione levels and the activity of enzymes associated with antioxidant protection in neonatal lung are increased in response to hyperoxia. Glutathione levels in developing rat lung decreased from 24 nmol/mg protein on day 19 of gestation to approximately 12 nmol/ mg protein at birth. The initial decrease in glutathione may be due to emergence of other antioxidant systems. Newborn rats placed in 100% oxygen showed a rapid and sustained increase in total glutathione levels which was primarily due to an increase in reduced glutathione. Explants obtained from 16-wk gestation human fetal lung or from 17- to 18-day fetal rat lung also showed increased total and reduced glutathione when cultured in 95% oxygen, 5% CO2as compared with explants cultured in room air. Type II cells isolated from neonatal rats maintained in oxygen for 6 days also showed glutathione levels twice those found in cells isolated from animals in room air. The activity of antioxidant enzymes (glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase) was increased in lungs of newborn rats exposed to 100% oxygen either at birth or 2 days of age. Antioxidant enzyme activity of lung explants cultured in 95% oxygen, 5% CO2was also higher than in explants maintained in room air. These results suggest that the increases in glutathione and of antioxidant enzymesin vivoandin vitroare a direct effect of oxygen exposure in lung and that the increase of both glutathione and antioxidant enzyme activity is intrinsic to the lung cell itself. It is likely that increases in glutathione in lung represent an important protective mechanism against oxidant injury.