The beneficial effects of supplemental oxygen delivered to patients suffering from acute respiratory distress is offset by its reduction to genotoxic reactive oxygen species (ROS) that inhibit proliferation and kill pulmonary cells. Cells respond to oxygen-induced damage by expressing the tumor suppressor p53 and the cyclin-dependent kinase inhibitor p21^{Cip1/WAF1/Sdi1} (p21), which limits proliferation by blocking entry into S phase. Since preventing DNA synthesis during genotoxic stress may enhance survival, the current study examines whether hyperoxia induces p21 through a p53-dependent pathway and whether p21 protects cells from the toxic effects of oxygen. HCT116 colon carcinoma cells and clonal lines lacking p53 or p21were used in this study because they allow direct cytotoxic comparisons between isogenic cells, without complications arising from unknown genetic differences between nonhomologous cell lines. Hyperoxia (95% O₂, 5% CO₂) increased p53 abundance, phosphorylation of p53 on serine 15, and p21 mRNA and protein in parental HCT116 cells that ceased proliferation. In contrast, p21 was not detected in either p53- or p21-deficient HCT116 cells, which exited the G1 compartment and were arrested in S and G2/M phases during hyperoxia. Trypan blue-dye exclusion revealed that induction of p21 markedly enhanced survival during exposure and colony survival assays showed that p21 enhanced the ability to resume proliferation during recovery in room air. The observation that p53-dependent induction of p21 prevents exit from G1 and promotes survival during hyperoxia is consistent with the importance of limiting DNA replication during genotoxic stress caused by oxygen exposure.