After DNA damage, many cells appear to enter a sustained arrest in the G(2) phase of the cell cycle. It is shown here that this arrest could be sustained only when p53 was present in the cell and capable of transcriptionally activating the cyclin-dependent kinase inhibitor p21. After disruption of either the p53 or the p21 gene, gamma radiated cells progressed into mitosis and exhibited a G(2) DNA content only because of a failure of cytokinesis. Thus, p53 and p21 appear to be essential for maintaining the G(2) checkpoint in human cells.