Expression of thymidylate synthase (TS) in tumor cells is frequently suggested as an important prognostic factor for patients scheduled for chemotherapy with 5-fluorouracil (5-FU). However, clinical evidence does not fully support such an anticipation. We studied the expression of rTSbeta, a reverse orientation gene of TS, as a 5-FU resistance marker in patients with primary breast cancer. Expression of rTSbeta was examined in 129 patients with newly diagnosed breast cancer and five breast cancer cell lines by immunohistochemistry, immunocytochemistry and immunoblotting. Clinically, expression of rTSbeta was found to correlate with survival of the patients (p=0.023) when patients received chemotherapeutic regimen containing 5-FU. In vitro, rTSbeta expression was found to correlate with 5-FU resistance in breast cancer cell lines. Notably, in the 5-FU-resistant cells, rTSbeta was identified in the nucleus, whereas in the 5-FU-sensitive cells, rTSbeta was found in the cytoplasm. Nuclear localization of rTSbeta was further found to be associated with protein farnesylation. Therefore, nuclear expression of rTSbeta could be a novel 5-FU resistance marker in patients with primary breast cancer.