Trifluorothymidine (TFT) is a fluorinated thymidine analog that after conversion to its monophosphate derivative can inhibit thymidylate synthase (TS) and be incorporated into DNA. TFT is a good substrate for thymidine phosphorylase (TP), and the combination of TFT and a TP inhibitor (TPI), called TAS-102, has been developed to enhance the bioavailability of TFT in vivo, and is currently being studied in a phase I study. We aimed to determine the limiting factor(s) in the cytotoxicity of TFT with or without TPI to cancer cells. Colon cancer and lung cancer cell lines with either an overexpression or deficiency of one of the enzymes involved in TFT metabolism were used to study the effect of TPI on TFT sensitivity and the role of TS inhibition. The synthesis of radioactive TFT metabolites was studied using thin-layer chromatography together with the incorporation of TFT into DNA. We found that despite a high rate of TFT phosphorolysis, cells with high TP expression are not more resistant to TFT, while TPI did not increase TFT sensitivity. High TS-expressing cells were shown to be cross-resistant to a 72-h exposure to TFT compared to 5-fluorouracil (5-FU), although this was more pronounced at a 4-h exposure (3.4-fold or more for TFT and 1.4-fold or more for 5-FU). Despite a moderate inhibition of TS activity in cells expressing high TS, these cells were more sensitive to TFT than 5-FU (3.8-fold or more). Only in Colo320TP1 cells expressing high TP, inhibition of TFT phosphorolysis by TPI increased formation of active TFT metabolites 1.8-fold, although this was not related to an increase in TFT incorporation into DNA. These studies show that uptake of TFT and subsequent phosphorylation of TFT by cancer cells is very rapid. Despite a high rate of degradation, the activation pathways are still saturated and sufficient to inhibit TS and enable incorporation into DNA, although the contribution of each effect is exposure time dependent.