In vivo hyperoxia exposure is a therapeutically relevant method for inducing lung injury that has been modeled in both rats and mice. In this study, C57Bl/6J mice were exposed to hyperoxia and allowed to recover in room air. The sub-lethal dose of hyperoxia for C57Bl/6J was 48 hours. Distal lung cellular isolates from treated animals were characterized as 98% epithelial, with minor fibroblast and endothelial cell contaminants. Cells were then verified as 95% pure AEC2 by Surfactant Protein-C (SP-C) expression. Following hyperoxia exposure in vivo, fresh, uncultured AEC2 were analyzed for proliferation by cell yield, cell cycle, PCNA expression and telomerase activity. DNA damage was assessed by TUNEL, while induction of DNA repair was evaluated by GADD 153 expression. A baseline level for proliferation and damage was observed in cells from control animals that did not alter significantly during acute hyperoxia exposure. However, a rise in these markers was observed 24 hours into recovery. Over 72 hours of recovery, markers for proliferation remained elevated, while those for DNA damage and repair peaked at 48 hours, then returned back to baseline. The expression of GADD 153 followed a distinct course, rising significantly during acute exposure and peaking at 48 hours recovery. These data demonstrate that in healthy, adult male C57Bl/6J mice, AEC2 proliferation, damage and repair follow separate courses during hyperoxia recovery and that both proliferation and efficient repair may be required to ensure AEC2 survival.