A panel of seven human colorectal cell lines of differing phenotype has been examined to elucidate the role of thymidylate synthase (TS) in the response to 5-fluoro-2'-deoxyuridine (FdUrd). Although TS is a major target of FdUrd, no consistent relationship was observed between the intracellular levels of TS and the response to FdUrd among the cell lines. Levels of thymidine kinase and dihydrofolate reductase, enzymes that are involved in generation of ligands that form the inhibitory ternary complex with TS, do not correlate with FdUrd response. Two cell lines that exhibit innate resistance to FdUrd, relative to the other cell lines, have variations in TS enzyme structure or gene structure. Cell line HCT 116 contains two forms of TS, as defined by isoelectric focusing. One form, which is unique to HCT 116, is more basic than the common form, which is present in all the cell lines. Cell line RCA contains a variation in the TS structural gene, as defined by restriction fragment-length analysis. These structural variations, which are associated with reduced response to FdUrd, may serve as markers for reduced clinical response to TS-directed chemotherapy.