Recently several advisory committees (EDSTAC, ICCVAM) have recommended that stable estrogen-dependent gene expression assays be developed for screening chemicals for estrogenic activity because of the high degree of specificity of the response and potential for use in a high-throughput mode. In this paper we describe a specific, sensitive assay developed for screening chemicals for estrogenic and antiestrogenic activities. T47D human breast cancer cells, which naturally express estrogen receptor (ER) alpha and beta, were stably transfected with a triplet ERE (estrogen-responsive elements)-promoter-luciferase reporter gene construct. The transformed cells were named T47D-KBluc. These cells are sensitive to the potent estrogens, 17beta-estradiol, ethynyl estradiol, and diethylstibesterol, and well-characterized weaker environmental estrogens like genistein, HPTE (an estrogenic pesticide metabolite), and 4-nonylphenol. The EC50 for estradiol was about 0.01 nM, reaching maximal induction at 0.1 nM. The antiestrogen, ICI 182,780, was able to completely inhibit the induction of luciferase expression by 0.1 nM estradiol at 10 nM, with an IC50 of 1 nM. In addition, we were able to replicate, in this in vitro assay, the observation that low concentrations of cadmium were able to induce estrogen-dependent gene expression, an effect that was completely inhibited by the potent antiestrogen ICI 182,780. The potent glucocorticoid receptor agonist, dexamethasone, was without effect as an ER agonist at concentrations up to 10 nM, whereas the potent androgen, dihydrotestosterone (DHT), showed no induction at concentration of 50 microM, but was a partial agonist at high concentrations of 0.2 mM and above. In summary, we have developed a specific, sensitive estrogen-responsive gene expression assay in a stable cell line that could possibly be adapted for high throughput screening of large numbers of chemicals for estrogenic and antiestrogenic activity. In addition, herein we also provide key protocol recommendations necessary to identify and eliminate common problems encountered in in vitro screening for estrogenicity.