5-Fluorouracil (5-FU) interferes with tumor-cell proliferation by inhibiting thymidylate synthase (TS). We examined the relationship between tandem repeat (TR) variations in the TS gene and survival following concurrent chemoradiotherapy in patients with esophageal squamous cell carcinoma (ESCC). TS-TR variations were analyzed in 57 stage II-IV ESCC patients undergoing chemoradiotherapy combined with 5-FU and cisplatinum (CDDP), and in 106 controls. Pretreatment non-neoplastic biopsy specimens from ESCC patients and lymphocytes from controls were used for analysis. Variations were identified by the size of DNA fragments amplified by polymerase chain reaction. Two to five TRs were found in Japanese individuals. TR3 homozygotes were predominant in 74% of ESCC patients and 61% of controls. Three-year survival rates were significantly longer in patients with TR2/2 or TR2/3 genotypes (38%) than in patients with TR3/3, 3/4, or 3/5 genotypes (9%; p=0.011). In the Cox proportional hazard model, the TR2/2 or TR2/3 genotypes were the only independent predictor for survival (Hazard ratio, 2.647; 95% confidence interval, 1.271-5.513). The TS-TR variations exert an important influence on survival following chemoradiotherapy in ESCC patients.