Abstract: Objective To investigate correlation between thymidylate synthase enhancer region (TSER) polymorphism and the antitumor activities of 5-fluorouracil (5-Fu) determined by ATP sensitivity assay in vitro. Methods The carcinoma cells isolated from fresh gastric tissue in 45 patients were cultured in vitro.The sensitivity of these cells to 5-Fu were tested using ATP-TCA method. TSER genotypes were detected by PCR and the correlation between different of TSER genotypes and its chemosensitivity to 5-Fu was analyzed in this study. Results In all cases, the frequency distribution of TSER 2R／2R、2R／3R and 3R／3R genotypes were 6.7%(3/45)、31.1%(14/45) and 62.2%(28/45), respectively. The overall effective rate of 3R/3R group to 5-Fu was 33.3%(14/42). In 2R／2R and 2R／3R genotype group its effective rate to 5-Fu was 56.3%(9/16), which was significantly higher than that of 3R／3R genotype group（19.2%，P<0.05). Conclusions The results in the present study suggest that the polymorphism of TSER was associated with the antitumor activities of 5 -fluorouracil, indicating that analysis of TSER polymorphisms might be useful to direct 5-fluorourcil-based antitumor chemotherapy.