Estrogen receptors ER␣ and ER␤ are members of the family of nuclear hormone receptors and act as ligandinducible transcriptional factors, which regulate the expression of target genes on binding to cognate response elements. We report here the characterization of steroid receptor coactivator-3 (SRC-3), a coactivator of nuclear receptor transcription that is a member of a family of steroid receptor coactivators that includes SRC-1 and transcription intermediate factor-2. SRC-3 enhanced ER␣ and progesterone receptor-stimulated gene transcription in a ligand-dependent manner, but stimulation of ER␤-mediated transcription was not observed. Protein-protein interaction assays, including real-time interaction analyses with BIAcore, demonstrated that the affinity of the ER␣ interaction with SRC-3 was much higher than that observed for the ER␤ interaction with SRC-3. Mutational analysis suggests a potential interplay between the transactivation function-1 and -2 domains of ER␣ and SRC-3. Furthermore, an intrinsic transactivation function was observed in the C-terminal half of SRC-3. Finally, SRC-3 was differentially expressed in various tissues and, among several tumor cells examined, was most abundant in the nuclear fraction of MCF-7 breast cancer cells. Therefore, SRC-3, a third member of a family of steroid receptor coactivators, has a distinct tissue distribution and intriguing selectivity between ER␣ and ER␤. Estradiol (E 2 )1 exerts numerous biological effects in different tissues through an interaction with the estrogen receptor (ER), a member of the steroid/nuclear hormone receptor superfamily (1, 2). Amino acid sequence analyses, transient transfection studies, and mutational dissections of ER indicate that ER can be subdivided into several functional domains (3). The N-terminal A/B domain contains a transactivation function, referred to as TAF-1. The DNA binding domain, the C region, contains two zinc fingers and is responsible for DNA recognition. The ligand binding domain (LBD) and a second transactivation function, referred to as TAF-2, is located at the C-terminal of ER. On binding to hormone, the receptor undergoes an activation and transformation step. The activated ER interacts with specific estrogen response elements that are located in the promoter region of estrogen-regulated genes and influences the rate of gene transcription. Over the past decade, numerous studies have provided a basic understanding of both the effects of ligand (agonist and antagonist) on the ER and the relationship between the structure and function of the ER (4). Nevertheless, little is known regarding the mechanisms involved in the gene-specific and tissue-selective effects mediated by either estrogens or antiestrogens. Furthermore, the molecular mechanisms by which ligand-activated ER influences the basal transcriptional machinery and regulates target gene transcription are mostly unknown.Recently, a new estrogen receptor, named ER␤, has been isolated from rat prostate and human testis (5, 6). The DNA binding domain of ER␤ is 9...