The lack of a phenotypic alteration of 5-hydroxymethyluracil (hmUra) DNA glycosylase (hmUDG) deficient Chinese hamster V79mut1 cells exposed to DNA-damaging agents known to produce hmUra has raised the question whether there might be DNA substrates other than hmUra for hmUDG. Based on the structural similarity between 5-chlorouracil (ClUra) and hmUra and the observations that 5-chloro-2'-deoxyuridine (CldUrd) induces base excision repair (BER) events, we asked whether hmUDG or some other DNA BER enzyme is responsible for the removal of ClUra from DNA. An in vivo flow cytometry assay with FITC-anti-BrdUrd (which cross-reacts with CldUrd) showed that exogenous CldUrd is incorporated into DNA. However, both in vivo and in vitro experiments indicated that ClUra is not excised from DNA by hmUDG or other DNA glycosylase activities. The absence of removal of ClUra by hmUDG raised the question whether DNA strand breaks occurred subsequent to thymidylate synthase inhibition, leading to deoxyuridine incorporation, followed by cleavage of uracil from DNA by uracil DNA glycosylase (UDG). An in vivo thymidylate synthase activity assay in V79 cells demonstrated that CldUrd treatment inhibits thymidylate synthase as effectively as 5-fluoro-2'-deoxyuridine (FdUrd) treatment. Uracil, a known UDG inhibitor, partially reverses the cytotoxic effects of CldUrd on V79 cells, thus confirming that CldUrd induced cytotoxicity is a result of UDG activity. Our results demonstrated that while CldUrd is not directly repaired from DNA, its cytotoxicity is directly due to the UDG removing uracil subsequent to inhibition of thymidylate synthase by CldUMP.