Trifluorothymidine (TFT) is well known to be converted to TFT-monophosphate by thymidine kinase and to inhibit thymidylate synthase. In addition, TFT-triphosphate (TFT-TP) is also incorporated into DNA, resulting in cytocidal effects. However, the precise mechanism of TFT-induced DNA damage is still unclear. Therefore, we investigated the modes of action of TFT against DNA replication and repair enzymes, as compared with those of 5FU and FdUrd. When HeLa cells were treated with TFT at a concentration of 1 µM (IC50 value), the concentration of TFT in the DNA was calculated as 62.2±0.9 pmol/1x106 cells for 4 h. On the other hand, following treatment of the cells with FdUrd (0.5 µM) and 5FU (10 µM) at their IC50 doses, the drug concentrations in the DNA were 7.53, and 0.17 pmol/1 x 10⁶ cells for 4 h, respectively. These results show the markedly greater degree of incorporation of TFT into the DNA of the HeLa cells compared with that of 5FU (approximately more than 300-fold for 4 h) or FdUrd (approximately more than 8-fold for 4 h). The primer extension assay demonstrated that TFT-TP was also incorporated into the T-sites of the growing DNA strand, however, it competed only weakly with thymidine triphosphate. The DNA glycosylase assay was performed using commercially available DNA glycosylase and fractionated HeLa cell extracts obtained by gel filtration. There was no detectable excision of the TFT pairing to adenine by uracil DNA glycosylase (UDG), thymine DNA glycosylase (TDG), methyl-CpG binding domain 4 (MBD4) or the fractionated HeLa cell extracts, however, TDG and MBD4 were able to excise the TFT pairing to guanine. Additional data indicate that small-interfering RNA-mediated knockdown of TDG or MBD4 significantly increased the resistance to the cytotoxic effects of FdUrd, but not to that of TFT. These studies show the greater degree of incorporation of TFT into the DNA than that of 5FU or FdUrd, and that such a high degree of incorporation of TFT residues into the DNA might be related to exhibit potent cytotoxic activity to be refractory to cleavage by these DNA glycosylases; thus, the DNA-directed cytotoxic effect of the compound is quite different from that of 5FU.