Uracil DNA glycosylase (UDG) is a base excision repair enzyme responsible for the removal of uracil present in DNA after cytosine deamination or misincorporation during replication. Inhibition of thymidylate synthase (TS), an important target for cancer chemotherapy, leads to deoxythymidine triphosphate (dTTP) pool depletion and elevation of deoxyuridine monophosphate (dUMP) pools which may also result in the accumulation of deoxyuridine triphosphate (dUTP). Large quantities of dUTP are believed to overwhelm the pyrophosphatase dUTPase, leading to misincorporation of uracil into DNA. Uracil is removed from DNA by uracil DNA glycosylase (UDG) resulting in an abasic site, but since the ratio dUTP:dTTP may remain high during continuing TS inhibition uracil can become re-incorporated into DNA causing a futile cycle eventually leading to DNA damage and cell death. This study has used isogenic cell lines differing in their expression of UDG to investigate the role of this enzyme in sensitivity to the specific TS inhibitors, ZD9331 and raltitrexed. The study showed that although increased expression and activity of UDG may lead to increased cell growth inhibition after TS inhibition over the first 24 h of treatment (measured using 3-(4,5-dimethyl (thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), probably due to increased damage to single-stranded DNA, the level of enzyme expression does not affect cell viability or cell death (measured using clonogenic assay, cell counting of attached/detached cells and cleavage of both poly ADP-ribose polymerase (PARP) and caspase 3). Increased expression and activity of UDG did not affect sensitivity to TS inhibition at later time points (up to 72 h treatment). Therefore UDG does not appear to play a major role in the response to TS inhibition, at least in the model used, and the results suggest that other determinants of response previously investigated, such as TS and dUTPase, may be more important for the response to TS inhibition.