Estrogen receptor (ER)-mediated gene transcription occurs via the formation of a multimeric complex including ligand-activated receptors and nuclear coactivators. We have developed a homogeneous in vitro functional assay to help study the ligand-dependent interaction of ERs with various nuclear coactivators. The assay consists of FLAG-tagged ER or ER ligand binding domain (LBD), a biotinylated coactivator peptide, europium (Eu)-labeled anti-FLAG antibody, and streptavidin-conjugated allophycocyanin (APC). Upon agonist binding, the biotinylated coactivator peptide is recruited to FLAG-tagged ER LBD to form a complex and thus allow fluorescence resonance energy transfer (FRET) to occur between Eu and APC. Compounds with estrogen antagonism block the agonist-mediated recruitment of a coactivator and prevent FRET. The assay was used to evaluate the preference of ERs for various coactivators and ligands. Both ER and ER exhibited strong preferences for coactivator peptides corresponding to SRC-1 and PGC-1 vs. PRIP and CBP. 17-estradiol (E2) acted as a non-selective agonist for ER and ER. Genistein showed full agonism for ER and only partial agonism for ER, but with higher potency for ER than ER. Both raloxifene and tamoxifen behaved as full antagonists in this functional assay. The results obtained using compounds with a wide range of potency correlated well with those from a cell-based reporter gene assay. Therefore, this simple in vitro functional assay is predictive of ligand-dependent transactivation function of the receptor and, as such, is useful in nuclear receptor applications including mechanistic studies.