A major mechanism underlying the cytotoxicity of fluoropyrimidine analogs such as 5-fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) occurs via the formation of 5-fluoro-2'-deoxyuridylate (FdUMP), a tight-binding inhibitor of thymidylate synthase (TS). Genetic variation in the structure of the TS molecule is an important determinant of response to fluoropyrimidines, because such variation may affect the binding of FdUMP to the enzyme. Previous studies have shown that the colonic tumor cell line HCT116 expresses two structurally distinct TS polypeptides that differ by the presence of tyrosine or histidine at residue 33. Compared with the Tyr-33 form, the His-33 form confers a 3-4-fold level of FdUrd resistance to cells; this was postulated to be derived from the reduced affinity of the enzyme for FdUMP and N5,N10-methylenetetrahydrofolate, ligands required for the formation of a stable inhibitory complex. In the present study, the Tyr-33 and His-33 forms have been purified to homogeneity, and their properties have been compared in detail. The Km values for dUMP and N5,N10-methylenetetrahydrofolate in the TS reaction were not significantly different between the two enzymes. In contrast, the catalytic efficiency (kcat) was 8-fold lower for the His-33 form. Kinetic and equilibrium binding measurements demonstrated that the dissociation constant for FdUMP binding into the ternary complex was 3-4-fold higher for the His-33 form; this was shown to be due to both a decrease in the rate of FdUMP association with the enzyme and an increase in the rate of FdUMP dissociation from the ternary complex. A TS form containing phenylalanine at residue 33 was created by site-directed mutagenesis and was shown to be very similar to the Tyr-33 enzyme with regard to kcat, pH/activity profile, and effect on FdUrd response. Thus, it is the presence of histidine at residue 33, rather than the absence of tyrosine, that is responsible for the alterations in catalytic and ligand-binding functions exhibited by the His-33 form. Possible mechanisms by which the histidine residue perturbs the structure of the TS active site are discussed.