Expression of estrogen receptor beta (ER-beta) and its regulation by estradiol and anti-estrogens was analyzed in breast cancer cells. We determined that ER-beta is expressed in normal and tumor human breast tissue as well as in breast cancer cell lines. We observed moderate levels of ER-beta expression in both T47D and T47D-V22 (a T47D variant cell line) cells, in contrast with T47DCo (a T47D variant cell line) cells when compared to ER-alpha expression. While T47DCo (a T47D variant cell line), BT474, MDA-MB-231, MDA-MB-453, MDA-MB-468 and MCF-7 express low levels of ER-beta, other cell lines including the T47D-Y (a T47D variant cell line), MDA-MB-435, BT-549, and SKBr-3 cells express undetectable levels of ER-beta. Interestingly, ER-beta and ER-alpha are apparently not co-expressed in the breast tissue analyzed. Estradiol induced 30-40-fold increased ER-beta mRNA expression in T47D cells over control untreated cells. Moreover, the anti-estrogen, 4-hydroxy-tamoxifen (4OH-Tam) strongly inhibited estradiol induction of ER-beta expression, but had little or no effect on estradiol induction of ER-alpha. A pure anti-estrogen, ICI-182,780, completely abolished the ability of estradiol to up-regulate the expression of ER. In addition, both actinomycin D and cyclohexymide inhibited estradiol induction of ER-beta mRNA, indicating that de novo mRNA and protein synthesis are probably required for this induction. In summary, this study demonstrates that ER-beta is expressed in breast cancer, and it is regulated by estradiol. Moreover, the studies demonstrate that estradiol up-regulation of ER-beta mRNA in T47D cells can be abolished by anti-estrogens. Thus, ER-beta expression may serve as a prognostic, diagnostic and/or therapeutic marker for breast cancer. To the best of our knowledge, this is the first report regarding hormonal regulation of ER-beta in human mammary cells.