It is now likely that the cyclin-kinase inhibitor, p21(WAF1/SD11), is a key effector of p53-mediated cell-cycle arrest at the G(1)/S checkpoint following DNA damage. More recently, however, in vitro data has suggested that this pathway may also mediate the acute inhibition of DNA synthesis seen in cells already in S phase. Here we address this question in an intact cell system using normal human diploid fibroblasts in which p53 function is manipulated by expression of a dominant-negative mutant (ala(143)) introduced by a retroviral vector. Induction of DNA strand breaks in normal control fibroblasts by exposure to bleomycin led as expected to G(1)/S cell cycle arrest, induction of p2l(WAF1) and a rapid reduction in the rate of DNA synthesis in cells already in S phase. Stable expression of mutant p53 abrogated the G(1)/S (but not the G(2)/M) cell cycle checkpoint and abolished the induction of p21(WAF1), but had no significant effect on the inhibition of DNA replication in S phase nuclei. We conclude that, despite the in vitro evidence for inhibitory activity on PCNA/polymerase delta, p21(WAF1) induction does not appear to be essential for the acute inhibition of DNA synthesis in the intact cell following strand-break damage in S phase.