Thymidylate synthase (TS, EC 2.1.1.45) is an important target enzyme for the fluoropyrimidines used in cancer chemotherapy. Studies have documented a 2- to 4-fold induction of TS protein following 5-fluorouracil (5-FU) treatment of malignant cells. We measured the effect that 5-FU exposure had on TS protein expression in nonmalignant human breast (MCF-10 and HBL-100), colorectal (ATCC Co18, Co112, and Co33), and bone marrow cells along with malignant breast (MCF-7) and colon (NCI-H630) cells. Twenty-four hours after plating, cells were treated with 0.01 to 10 microM of 5-FU for a period of 24 hr. TS was quantitated by Western immunoblot using monoclonal antibody TS106. Absolute levels of TS in nonmalignant cells were substantially lower than in the malignant lines, ranging from approximately 40% in HBL-100 cells to less than 10% in the colon lines. An approximately two-fold induction in the level of TS was found for all cell lines examined, and there was a strong dependence on 5-FU exposure concentration in free TS levels of MCF-WT, and total TS levels of H630-WT, normal bone marrow, and MCF-10 cells. The induction of TS following 5-FU exposure is a generally observed phenomenon in both malignant and nonmalignant cells, suggesting that a selective means for inhibiting this induction may be critical for the development of alternative therapeutic strategies using 5-FU and the antifolate TS inhibitors.