Exposure of 21-day-old Sprague-Dawley rats to hyperoxia (> 95% O2 for 8 days) causes thickening of the airway epithelial and smooth muscle layers. To test the hypothesis that hyperoxic exposure increases airway layer DNA synthesis, we labeled the nuclei of cells undergoing S-phase by administering the thymidine analog bromodeoxyuridine (BrdU). BrdU was administered on days 3 and 4, 5 and 6, or 7 and 8 of air or O2 exposure, and the lungs were harvested immediately thereafter. Histologic sections were stained with an avidin-biotin-immunoperoxidase stain that revealed BrdU incorporation into nuclei, and a hematoxylin counterstain. After 4 days of air or O2 exposure, there was no difference in BrdU fractional labeling between control and hyperoxic animals. Thereafter, fractional BrdU labeling of the small airway (circumference < 1,000 microns) epithelium and smooth muscle layer was significantly increased in O2-exposed animals (P < 0.01, unpaired t test). The fractional labeling of larger, central airway smooth muscle layer cells was also increased after 8 days of O2 exposure (P < 0.01). In another cohort of O2-exposed animals, measurements of airway layer dimensions demonstrated increases in small airway epithelial and smooth muscle layer thickness that paralleled the time course seen for BrdU incorporation. We conclude that O2 exposure of immature rats increases airway epithelial and smooth muscle layer cellular DNA synthesis. These data suggest that hyperplasia of airway epithelial and smooth muscle layer cells may contribute to hyperoxia-induced airway remodeling.