In a prior National Surgical Adjuvant Breast and Bowel Project (NSABP) adjuvant study, the addition of the antiestrogen tamoxifen to chemotherapy with melphalan and fluorouracil adversely affected survival in several patient subsets, suggesting an antagonistic drug interaction. To investigate this possibility, we studied the interaction of tamoxifen and other antiestrogens with several cytotoxic drugs in cultured human breast cancer cell lines. Clinically relevant concentrations of tamoxifen and melphalan reduced colony survival of estrogen receptor (ER)-positive breast cancer cells when used alone in a colony-forming assay. However, pretreatment of cells with tamoxifen followed by exposure to melphalan resulted in antagonism, with more colonies surviving treatment with the combination than with melphalan alone. Identical effects were seen using several other triphenylethelene antiestrogens. An antagonistic interaction was observed even with a brief preincubation with tamoxifen that had no effect on cell proliferation, indicating that antagonism was not due to tamoxifen's known cell kinetic effects. Tamoxifen even antagonized melphalan cytotoxicity in ER-negative breast cancer cells and in cultured liver cells. An additive drug interaction occurred when melphalan was combined with pharmacologic concentrations of estradiol or medroxyprogesterone acetate, but antagonism was also observed with dexamethasone. Tamoxifen also antagonized the cytotoxicity of fluorouracil in these cells. However, an additive interaction occurred when the antiestrogen was combined with doxorubicin or 4-hydroxy-cyclophosphamide, an alkylating agent that is transported into the cell by a different carrier-mediated mechanism than melphalan. To avoid potential antagonism in the clinic, combinations of tamoxifen with melphalan and/or fluorouracil should be avoided.