Two human colorectal tumor cell lines are differentially sensitive to growth inhibition by 5-fluorodeoxyuridine (FdUrd); cell line RCA is less sensitive to FdUrd than is cell line C. Thymidylate synthase (TS), a target of FdUrd, has been purified to homogeneity from both cell lines. Because of differences in the avidity for a folate ligand affinity matrix, TS forms from the cells were purified by two different procedures. Relative to the enzyme from C cells, the enzyme from RCA cells demonstrated higher Km values for the substrates deoxyuridylate and 5,10-methylene-tetrahydrofolate, a lower rate of association of the inhibitor 5-fluorodeoxyuridylate (FdUMP), a similar rate of FdUMP dissociation, and lower enhancement of covalent FdUMP binding by folate derivatives. The activities of the enzymes in situ and the catalytic efficiencies of the purified enzymes were similar. Thus, a cell line that is naturally resistant to FdUrd has been identified that expresses a TS with reduced affinity for FdUMP and 5,10-methylenetetrahydrofolate, relative to the enzyme expressed in a FdUrd-sensitive cell line.