Abstract: Objective To investigate the mechanism of acquired resistance of pancreatic cancer cell Puta8988 to pemetrexed and to analyze its correlation with the expression of TS,FPGS and RFC.Methods After more than two months inducing of pemetrexed,the cytotoxicity of drugs to the parental and resistant lines was assessed by MTT assay.When the IC5 of the resistant lines raise to a level which our experiment need,it is considered acquiring the character to resistance pemetrexed.Then the mRNA expression levels of TS,FPGS and RFC were detected using RT-PCR.Results Pancreatic cancer cells acquired drug-resistance to pemetrexed.After recurrent treatment with pemetrexed,the IC₅₀ values in Puta8988 cells were increased by 2.2 fold as compared to their parental cells.(P ＜ 0.05).RT-PCR showed a correlation between TS,FPGS and RFC mRNA expression levels and the sensitivity to pemetrexed after pemetrexed treatment.Conclusion These findings reveal that the levels of TS,FPGS and RFC following recurrent exposure to pemetrexed are associated with resistance to these drugs.These findings suggest that the activation of TS,FPGS and RFC genes after recurrent drug exposure contributes to the chemoresistance of pancreatic cancer cells and that their intervention might enhance chemosensitivity in pancreatic cancer.