5-Fluorouracil (5FU), an antimetabolite often used for the treatment of breast cancer, binds to and inactivates the enzyme thymidylate synthase (TS). Measurement of TS levels may be useful in examining resistance to 5FU, but current methods involving ligand binding assays present considerable problems due to the low basal level of this enzyme in many tissues. Therefore, a semiquantitative polymerase chain reaction (PCR) technique to measure TS RNA concentration in breast tumour cell lines has been developed with potential for measuring TS RNA levels in individual breast tumours. Using less than 1 microgram of total RNA, we determined that the T47D breast cancer cell line expressed TS levels 1.3 to 4.4 times lower than those of four other breast cancer cell lines, MCF-7, MDA-MB-468, MDA-MB-231, and MDA-A1R. Using the MCF-7 cell line as a representative of relatively high TS expression, the relationship between the S-phase fraction (SPF), and TS levels in MCF-7 and T47D cells was next examined because cell cycle kinetics can also influence drug response. TS expression was directly related to SPF. MCF-7 cells had a higher SPF, corresponding to their higher TS level relative to T47D cells. Lastly, 5FU sensitivity was assayed for and it was found that MCF-7 cells were more sensitive to 5FU than T47D, despite having higher TS levels. A semiquantitative technique for assessing TS RNA expression by PCR in breast cancer cell lines has been demonstrated. In the cell lines tested, there was no direct relationship between TS level and 5FU sensitivity, though there was a direct relationship between cell proliferation and sensitivity to 5FU.