BACKGROUND Currently, one of the most effective strategies for the treatment and prevention of breast cancer is the use of drugs that block estrogen action in the breast. The success of the first clinically relevant selective estrogen receptor modulator (SERM), tamoxifen, provided the foundation for further testing of this drug to reduce breast cancer incidence in high-risk women. However, the negative effects associated with the long-term use of tanrhoxifen have initiated the search for compounds that are more effective but less toxic. The discovery of raloxifene (RAL), which functions as a potent antiestrogen in the breast but an estrogen receptor (ER) agonist in the bone and cardiovascular system with very little uterotropic activity, provided an alternative strategy to the targeted use of tamoxifen. The aim of this study was to evaluate RAL in combination with 5-fluorouracil (5-FU)/trimetrexate (TMX) to determine the most effective regimes and cellular mechanism of action to mitigate trimetrexate cytotoxicity in human bone marrow cells. METHODS The cell viability was performed using the Quick Cell Proliferation Assay by exposing the cells to TMX, 5-FU and RAL alone; RAL 24 h prior to 5-FU followed 2 h by TMX, and 5-FU 2 h prior to TMX followed 24 h by RAL determined the sequence-dependent interaction between TMX, 5-FU and RAL on the proliferation. RESULTS The growth rate in MCF-7 in late RAL was 34.75 +/- 4.79% of the control, whereas in bone marrow the same drug combination exhibits a significant protection against TMX cytotoxicity with late RAL yielding 51.25 +/- 4.43% of the control. The findings were also supported by Cell flow cytometry and Western blot analysis. CONCLUSIONS Sequence-dependent administration of RAL in combination with 5-FU/TMX can act against TMX toxicity in human bone marrow, while not affecting the maximum inhibitory effect of TMX in breast cancer.