Estrogen receptor (ER), one member of nuclear hormone receptor (NR) family, is an estrogen-dependent transcriptional factor that plays an important role in development, progression and treatment of breast cancer. Transcriptional co-factors (co-activators and co-repressors) are critical for ER to transduce hormone and metabolic signaling to target genes. A number of functional and structural studies have elucidated the precise mechanisms of co-activator interaction with the ligand-inducible activation domain in ER via one and several LXXLL motifs (where X is any amino acid) known as NR-Box. By the yeast two-hybrid system we have identified a novel ER-a interacting protein ERIAP (Estrogen Receptor Interacting and Activating Protein) which contains two consensus LXXLL motifs. ERIAP associated with ER-a in a ligand-dependent manner, as demonstrated by in vivo immunoprecipitation and in vitro GST capture assays. The two NR boxes were essential for ERIAP interaction with ER-a. Furthermore, ERIAP specifically enhanced ligand-mediated ER-a transcriptional activity in a dose-dependent fasion and increased the expression of estrogen-responsive gene pS2. Thus, our present findings indicate that ERIAP funcions as a new coactivator for ER-a transcriptional activity, which may play an important role in development and progression of breast cancer. Estrogen receptor (ER), one member of nuclear hormone receptor (NR) family, is an estrogen-dependent transcriptional factor that plays an important role in development, progression and treatment of breast cancer. Transcriptional co-factors (co-activators and co- repressors) are critical for ER to transduce hormone and metabolic signaling to target genes. A number of functional and structural studies have elucidated the precise mechanisms of co-activator interaction with the ligand-inducible activation domain in ER via one and several LXXLL motifs (where By the yeast two-hybrid system we have identified a novel ER-a interacting protein ERIAP (Estrogen Receptor Interacting and Activating Protein) which contains two consensus LXXLL motifs. ERIAP associated with ER- a in a ligand-dependent manner, as demonstrated by in vivo immunoprecipitation and in vitro GST capture assays. The two NR boxes were essential for ERIAP interaction with ER-a. Furthermore, ERIAP specifically enhanced ligand-mediated ER-a transcriptional activity in a dose-dependent fasion and increased the expression of estrogen-responsive gene pS2. Thus, our present findings indicate that that ERIAP funcions as a new coactivator for ER-a transcriptional activity, which may play an important role in development and progression of breast cancer.