This article describes the development and validation of two fluorescent receptor assays for the hRec-estrogen receptor subtypes alpha and beta. As a labelled ligand an autofluorescent phyto-estrogen (coumestrol) has been used. The estrogen receptor (ER) belongs to the nuclear receptor family, a class of soluble DNA binding proteins, mainly present in the cytoplasm of the cell, that act as ligand-activated enhancer factors. It consists of two different forms, expressed as ER-alpha (66 kDa) and ER-beta (59 kDa). The ER-alpha is mainly located in the uterus and the ER-beta can be found in vascular tissue. Detection and identification of compounds having estrogenic effects is of importance in drug discovery programmes within the pharmaceutical industry for their search for ER-subtype selective (ant)agonists which may prove to be of therapeutic value in treating a variety of estrogen-linked pathologies (breast cancer, osteoporosis, cardiovascular disease, type II diabetes and Alzheimer disease). Furthermore, interactions of (xeno-)estrogens with the endogenous hormonal system of the exposed organism can affect embryos, gonads, and reproductive behaviour. The latter can eventually lead to reduced reproduction and deterioration of a population. For that reason, monitoring of (xeno-)estrogens in food products and in the environment, attracts considerable attention by health councils throughout the world. The following characteristics were obtained for the human recombinant (hRec) estrogen receptor-beta assay, which is suitable for ER subtype selective drug-discovery purposes (IC50 values for 17-beta-estradiol and genistein were 5.1 nM and 25 nM, respectively): goodness of fit (R2) was always > 0.98 (x = 0.9933, n = 10). LLOQ of the assay is typically > or = 500 picomolar, whereas the ULOQ of the assay is < or = 20.0 nanomolar. For the hRec-estrogen receptor-alpha assay, which is suitable for monitoring of (xeno-)estrogens (IC50 values for 17-beta-estradiol and genistein were 0.68 nM and 65 nM, respectively) the following characteristics were obtained: goodness of fit (R2) was always > 0.96 (x = 0.9838, n = 10). LLOQ of the assay is typically > or = 200 picomolar, whereas the ULOQ of the assay is < or = 5.0 nanomolar.