Deoxyuridine triphosphate (dUTP) misincorporation and uracil misrepair have long been implicated in fluoropyrimidine-induced DNA damage; however, the enzymatic activities responsible for these lesions have not been previously identified as critical determinants of overall sensitivity to the antitumor effects of these agents. The purpose of this study was to determine whether differences in uracil misincorporation/misrepair could account for the difference in sensitivity to fluorodeoxyuridine (FdUrd)-induced cytotoxicity and DNA damage in 2 human colorectal tumor cell lines having identical sensitivities to FdUrd-induced thymidylate synthase inhibition. Compared to HT29 cells, SW620 cells were resistant to both cytotoxicity and induction of DNA double-strand breaks, as assessed by pulse field gel electrophoresis. Alkaline elution experiments demonstrated that this resistance coincided with delayed induction of DNA single-strand breaks on parental DNA and, to a lesser extent, on nascent DNA. Following treatment with FdUrd for 24 h, HT29 cells accumulated 904 +/- 273 pmol deoxyuridine triphosphate (dUTP)/10(7) cells, whereas SW620 cells accumulated 20 +/- 7 pmol dUTP. Consistent with this difference in extent of dUTP accumulation was the observation that deoxyuridine triphosphatase levels in SW620 cellular extracts were 4.4-fold higher than in HT29 extracts. The ability to accumulate dUTP, intracellular deoxyuridine triphosphatase activity, and extent of DNA damage appear to be important determinants for predicting the response to FdUrd treatment in these cell lines.