We have examined the effects of conditionally expressing wild-type p53 activity in HT29 cells on DNA damage and cytotoxicity caused by exposure to fluorodeoxyuridine (FdUrd). Expression of wild-type p53 phenotype for 24 hr before FdUrd treatment provided HT29 cells with virtually complete protection from cytotoxicity caused by this drug. In addition, wild-type p53 expression also prevented FdUrd-induced DNA double-strand breaks and, unexpectedly, single-strand breaks in parental (mature) DNA. Temporary expression of wild-type p53 activity in the absence of drug treatment caused some loss of clonogenicity, although the magnitude of this cytotoxic effect was small compared with the level of cell kill obtained by treatment with cytotoxic drugs for similar periods of time, indicating that HT29 cells are not highly sensitive to induction of programmed cell death by wild-type p53. Because these observations conflict with previously suggested models for FdUrd-induced damage to parental DNA, we propose an alternative model to explain how incorporation of uracil into nascent DNA might result in single-strand breaks in the opposite (parental) strand and how these breaks might be converted to the double-strand breaks that produce cell death.