Thymidylate synthase (TS) is an important target for chemotherapy drugs, such as 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (FUDR), oral 5-FU prodrugs (e.g., uracil/tegafur [UFT], S-1, and capecitabine), and other novel folate-based drugs (e.g., raltitrexed, pemetrexed, and nolatrexed). Overexpression of TS is linked to resistance to TS-targeted chemotherapy drugs. Polymorphic tandem repeats located in the TS enhancer region (TSER) have been shown to influence TS expression. Three copies (TSER*3) of the tandem repeat give a 2.6-fold greater in vitro TS expression than 2 copies (TSER*2). A stepwise increase in expression and enzyme activity has been observed with increasing copies of the TSER. Alleles containing 4 (TSER*4), 5 (TSER*5), and 9 (TSER*9) copies of the tandem repeat have also been identified, although the effect of these alleles remains unclear. Preliminary data have suggested that stage III colorectal cancer patients with the TSER*3/TSER*3 genotype do not receive the improvement in survival from adjuvant 5-FU observed in patients with the TSER*3/TSER*2 or TSER*2/TSER*2 genotype. A poor response rate to 5-FU for neoadjuvant rectal or metastatic colorectal disease is also apparent in TSER*3/TSER*3 patients. This has significant clinical implications because TSER*3/TSER*3 occurs in 30% of Caucasian patients. Ethnic variation also exists in the TSER, with Asian populations having significantly higher frequency of TSER*3 than other world populations. Prospective confirmation of the impact of TSER on outcome, after TS-targeted chemotherapy, will define the utility of pharmacogenetics to optimize the selection of 5-FU therapy for colorectal cancer.