The present study investigated the effect of the phytochemical genistein on the proliferation and differentiation of MCF-7 and 3T3-L1 cells via the regulation of estrogen receptor-alpha (ER alpha) expression and the induction of apoptosis. When MCF-7 human breast cancer cells were treated with 50, 100, 150 and 200 mu M genistein for 24,48 or 72 h, cell growth was significantly decreased in a concentration-dependent manner. Notably, the patterns of ERa expression and proliferation in MCF-7 cells treated with genistein were similar. Furthermore, ER alpha expression in differentiating 3T3-L1 cells was significantly inhibited by 48 h treatment with 50 mu M genistein, which was selected based on the results of cytotoxicity assays on 3T3-L1 preadipocytes [lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays]. Under the same conditions, genistein-induced apoptotic features were observed in MCF-7 and differentiating 3T3-L1 cells. This observation is supported by the finding that B-cell lymphoma 2 (Bcl-2) expression was reduced while that of Bcl-2-associated X protein (Bax) was induced by genistein. The results of the present study suggest that an ER alpha-related pathway and the induction of apoptosis are involved in the proliferation of MCF-7 cells and the differentiation of 3T3-L1 cells.