Coumestrol is a well-known ligand for the estrogen receptor (ER). The compound itself is fluorescent, and its fluorescence intensity at 408 nm increases upon binding to the ER. Here we describe a novel binding assay in 96-well plate format for estrogenic compounds, based on the competition between fluorescent coumestrol, and estrogenic compounds for binding to the ligand binding domain (LBD) of the ER-alpha. Displacement of coumestrol was measured as a decrease in fluorescence intensity using a Victor-1420 multilabel reader. Competitive binding curves for the well-known estrogenic Compounds, 17 beta-estradiol (E-2), ethinylestradiol, 4-nonylphenol, 4-octylphenol, genistein, bisphenol A, tamoxifen and diethyl-stilbestrol were constructed by using 7-10 different concentrations of the compounds and a fixed concentration of ER-alpha-LBD (14 nmol) and coumestrol (100 nmol). IC50) values and relative potencies (compared to E-2) of the estrogenic compounds were determined. The assay was validated by comparing the relative potencies to those from standard radioligand binding assays in the literature. Within day and between day variations were determined and the performance of the assay was assessed by determining the coefficients of variation and Z ' values. The present fluorescent binding assay has proven to be fast and easy, and allows accurately quantifying the binding affinity of estrogenic ligands. The method is also suitable as a high-throughput screening assay for ER ligands.