Tamoxifen (TAM), the non-steroidal anti-estrogen most widely administered to breast cancer patients, acts, at least in part, by competing with estrogen receptors (ER). However, the existence of an alternative mechanism of action for this drug is supported by the clinical observations that: (a) 30% of patients with ER-negative cancer cells respond to TAM, and (b) 30% of patients with ER-positive cancer cells are not sensitive to this anti-estrogen. In this study, we observed that growth of the human ER-negative breast cancer cell line MDA-MB-435 was inhibited by TAM and 4-hydroxytamoxifen (4OH-TAM) in a concentration-dependent fashion. Both monoclonal enzymoimmunoassay and Dextran Charcoal Coated Scatchard radioimmunoassay analysis demonstrated that this MDA-MB-435 cell line does not express ER. The absence of ER in MDA-MB-435 cells was also demonstrated at the mRNA level by both northern blot hybridization and reverse transcription-polymerase chain reaction techniques. MDA-MB-435 cell proliferation was not affected by 17 beta-estradiol or by the pure anti-estrogen ICI 164384, further demonstrating that the observed effects of TAM and its active metabolite on the proliferation of MDA-MB-435 cells were due to an ER-independent mechanism, yet to be identified. MDA-MB-435 thus appears to be a promising original model for the study of the alternative ER-independent mechanisms of action of TAM.