In hormone-dependent tissues such as breast and ovary, tumorigenesis is associated with an altered expression ratio between the two estrogen receptor (ER) subtypes. In this study, we investigated the effects of ERbeta ectopic expression on 17beta-estradiol (E2)-induced transactivation and cell proliferation in ERalpha-positive BG1 ovarian cancer cells. As expected, ERbeta expression strongly decreased the mitogenic effect of E2, significantly reduced E2-dependent transcriptional responses (both on a stably integrated estrogen response element [ERE] reporter gene and on E2-induced mRNAs), and strongly enhanced the formation of ER heterodimers as evidenced by chromatin immunoprecipitation analysis. Inhibition by the ERalpha-selective ligand propyl pyrazole triol was less marked than with the pan-agonist (E2) or the ERbeta-selective (8beta-vinyl-estradiol) ligands, indicating that ERbeta activation reinforced the inhibitory effects of ERbeta. Interestingly, in E2-stimulated BG1 cells, ERbeta was more efficient than ERalpha to regulate the expression of receptor-interacting protein 140 (RIP140), a major ERalpha transcriptional corepressor. In addition, we found that the RIP140 protein interacted better with ERbeta than with ERalpha (both in vitro and in intact cells by fluorescence cross-correlation spectroscopy). Moreover, RIP140 recruitment on the stably integrated reporter ERE was increased upon ERbeta overexpression, and ERbeta activity was more sensitive to repression by RIP140. Finally, small interfering RNA-mediated knockdown of RIP140 expression abolished the repressive effect exerted by activated ERbeta on the regulation of ERE-controlled transcription by estrogens. Altogether, these data demonstrate the inhibitory effects of ERbeta on estrogen signaling in ovarian cancer cells and the key role that RIP140 plays in this phenomenon.