We cloned the cDNA of a novel steroid receptor-binding protein, SRB-RGS, which suppressed the estrogen receptor (ER)alpha-mediated and other promoter-driven transcriptional activities. This study revealed the interaction between the full-length SRB-RGS and full-length ERalpha or ERbeta by a coimmunoprecipitation assay. The full-length SRB-RGS and full-length ERalpha interacted in COS-7 cell by a mammalian two-hybrid system. The interaction between intrinsic SRB-RGS and ERs in the nuclear ER extract from the rat uteri was observed by the gel-shift assay. These results strongly suggested that SRB-RGS interacts with ERs bound to DNA (estrogen response element) in the nuclei of the cells. SRB-RGS suppressed very efficiently the ERalpha-, ERbeta-, and ERalpha+ERbeta-mediated transcriptional activities. Green fluorescence of enhanced green fluorescence protein (EGFP)-tagged SRB-RGS was localized both in the nucleus and in the cytoplasm. Intrinsic SRB-RGS was immunostained in the nucleus and the cytoplasm of HeLa cells. The putative SRB-RGS deduced from cDNA sequence was identified by the immunostaining and Western blotting by using the anti-SRB-RGS antibody. Overexpression of SRB-RGS induced the cell death in the HeLa cells. The nucleotide sequence of SRB-RGS cDNA that we cloned previously is identical with that of the newly isolated RGS3 cDNA. SRB-RGS could interact with ERs bound DNA in the nuclei of the cells and suppressed the ERs-mediated transcriptional activities.