A panel of human colorectal tumor cell lines has been examined to determine the role of TS in the response to fluoropyrimidine antimetabolites. Among these cell lines, the response to FdUrd does not correlate with the levels of TS. In cell lines HCT 116 and RCA, which are poorly responsive to FdUrd, structural alterations in TS have been identified. In HCT 116, two TS polypeptides are present: a common form, occurring in all the cell lines and a variant form. The variant TS polypeptide has a reduced affinity for the TS ligands, FdUMP and CH2H4PteGlu, relative to the common TS polypeptide. Clonal populations of HCT 116 that overproduce each form have been isolated. Clones that overproduce the variant polypeptide are 4-fold less responsive to TS-directed cytotoxic agents than those that overproduce the common; thus, the presence of the variant TS is associated with a reduced response to TS-directed cytotoxic agents. The response of cell line RCA to FdUrd is dependent upon the extracellular CF concentration: response increases as CF is increased. RCA contains a TS enzyme with reduced affinity for CH2H4PteGlu, relative to cell line C, which is sensitive to FdUrd at all CF concentrations. Both cells form high chain-length polyglutamates of CH2H4PteGlu at CF concentrations in which the response to FdUrd differs by 4-fold. In RCA, the TS structural gene is variant, relative to the other cell lines. This variation may underlie the altered enzyme affinity for CH2H4PteGlu and the sensitivity to modulation of FdUrd response by CF.