We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.