We hypothesized that a possible mechanism to explain the significant increases that occur in the pulmonary antioxidant enzyme (AOE) system late in gestation might be an endogenous increase in the normal reactive O2 substrates for these enzymes. We found that lung O2 free radical formation increased approximately 175% between fetal day 18 and birth (p < 0.01). We also found that late fetal rat lung mitochondrial and microsomal rates of AOE substrate (H2O2) generation increased markedly, and there was also significantly increased lung lipid peroxidation products with increasing gestational age. These definite elevations in reactive O2 species production in parallel with the time course of maturational elevations in the pulmonary AOE system, suggest that increasing enzyme substrate concentrations could be a primary controlling mechanism for increasing lung AOE gene expression in preparation for birth of the newborn.