Abstract: Objective To study the dynamic changes in thymidylate synthase (TS) expression in different cell lines treated with nolatrexed. Methods For studying the time course of the action of the drug, LoVo and Lo2 cells were treated separately with nolatrexed at the concentrations of 10 and 75 γmol/L (approximately their respective IC₉₀ value) for 0 to 60 h, while in the dose response study, treatment with nolatrexed for 36 h was performed with the dose range of 0 to 40 γmol/L for LoVo cells and 0 to 125 γmol/L for Lo2 cell. TS expression was detected by flow cytometry and Western blotting. Results The dynamic changes in TS expression with respect of the time course and dose response differed significantly in the two cell lines after nolatrexed treatment. In the time course study, TS expression increased continuously in LoVo cells, as compared with that in the control cells, whereas the TS expression initially decreased and then began to increase gradually 24 h after the treatment, remaining still below the control level after 60 h. In the dose response study, TS expression in LoVo cells gave rise to a peak in the curve, with the peak level approaching the IC₉₀ value. In Lo2 cells, when the concentration of nolatrexed was blow the IC₉₀ value, the decrement in TS expression were similar, but as the concentration exceeded the IC₉₀ value, TS expression decreased dose-dependently. Conclusion Nolatrexed may elicit TS induction in the cells, and the difference in the dynamic changes of TS expression between different cell lines after nolatrexed treatment contributes to different profiles of drug resistance.