In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERα) in an agonist-dependent manner. The interaction is specific for ERα as ERβ and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERα was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERα is sufficient for interaction with RAP80. RAP80 overexpression reduces ERα polyubiquitination, increases the level of ERα protein, and enhances ERα-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ERα protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UIMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UIMs does not affect the ability of RAP80 to interact with ERα, but eliminates the effects of RAP80 on ERα polyubiquitination, the level of ERα protein, and ERα-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ERα as a new RAP80-interacting protein and suggests that RAP80 may be an important modulator of ERα activity. © 2007 Oxford University Press.