Thymidylate synthase (TS) is a target enzyme of 5-fluorouracil (5-FU), and the TS gene has a polymorphic tandem repeated sequence in the 5'-untranslated region. We investigated the polymorphism on the repeat length of the TS gene and its relation to the number of 5-fluoro-dUMP (FdUMP) binding sites in human gastrointestinal cancers. Seventy adenocarcinoma samples were used. Twenty-one patients received UFT and 49 patients received no treatment before surgery. Tandem repeat length of TS gene was determined by PCR amplification of genomic DNA. The number of FdUMP binding sites were measured by 3H-FdUMP binding assay. Double- (2R) and triple-repeated (3R) sequences of the TS gene were found in the cancer tissues. Three genotypes of TS were found: 2R/2R (n = 3), 2R/3R (n = 16) and 3R/3R (n = 51). The free TS level for the 3R/3R genotype was significantly higher than for the 2R/3R genotype in UFT (+) group. Similar genotype-dependent difference of TS level was observed in UFT (-) group. Our results suggest the link between TS genotype and TS protein expression. This link might offer an advantage for selection of patients to receive 5-FU-based chemotherapy.