Nucleic acid-metabolizing enzymes, such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT), have attracted attention as candidates for response determinants of 5-fluorouracil (5-FU). Whether the expression levels of these enzymes can be adopted as valuable parameters for 5-FU sensitivity in breast cancer has yet to be elucidated. In the present study, intratumoral mRNA expression of TS, DPD, TP and OPRT were determined in formalin-fixed paraffin-embedded surgical specimens collected from 217 breast cancer patients, using the Danenberg Tumor Profile method, which combines microdissection and real-time-polymerase chain reaction. The significance of these enzymes as prognostic and 5-FU efficacy-predicting factors was evaluated. Our data showed that a low DPD expression is related to a high nuclear grade and other factors including hormone receptor-negativity. Low expression levels of TP were found in hormone receptor-negative tumors. TS and OPRT expression were not related to various clinicopathological factors, but patients with a high TS mRNA expression showed a significantly poorer prognosis in cases where 5-FU was not administered. The efficacy of 5-FU was more significant when administered for more than 6 months in the group with a high TS mRNA expression. These data suggest that TS mRNA expression in breast cancer tissue is an ideal predictor of outcomes for patients with no administration of 5-FU, and of the efficacy of 5-FU.