The enzyme thymidylate synthase (TS) is an important target of 5-fluorouracil (FUra) that is utilized for the treatment of disseminated colorectal cancer. One determinant of clinical response to FUra-based therapy is TS expression. with high levels of expression being predictive of poor response. In the present investigation the levels of immunoreactive TS were analyzed in human colon metastases in the liver (n=l1). The levels of TS ranged from 0.30 to 4.60 pmol TS/g tissue. A good correlation was observed between the levels of immunoreactive TS and TS mRNA (n=6, r=0.69). Of the 11 metastases analyzed, 5 exhibited relatively high levels of TS expression. Two metastases with high TS expression were obtained from patients who received adjuvant therapy with FUra. In 4 metastases with relatively high levels of TS expression, TS gene copy number was analyzed. No evidence for amplification of TS gene sequences was observed. The basis for the high levels of TS expression was examined by structural analysis of TS cDNA. No nucleotide sequence differences were detected in the coding regions of the TS genes from the metastases. Mutations were detected at positions 961 and/or 1031 in the 3'-untranslated regions of the TS gene from the metastases; mutations at these sites were also detected in DNA isolated from normal colon mucosa (n=4) and primary colorectal tumors (n=4). No correlation was observed between TS expression and the nucleotide alterations at these positions. Polymorphism was observed in the 5'-untranslated regions of the TS gene in hepatic metastases (n=6). A general trend was observed between the structure of the 5'-untranslated region of the gene and TS expression.