Cellular levels of estrogen receptor-alpha (ER) protein are regulated primarily by the ubiquitin-proteasome pathway. Dynamic interactions between ER and the protein degradation machinery facilitate the downregulation process by targeting receptor lysine residues for polyubiquitination. To date, the lysines that control receptor degradation have not been identified. Two receptor lysines, K302 and K303, located in the hinge-region of ER, serve multiple regulatory functions, and we examined whether these might also regulate receptor polyubiquitination, turnover, and receptor-protein interactions. We utilized ER-negative breast cancer C4–12 cells to generate cells stably expressing wild-type (wt) ER or ER with lysine-to-alanine substitutions at K302 and K303 ("ER-AA"). In the unliganded state, ER-AA displayed rapid polyubiquitination and enhanced basal turnover, as compared to wtER, due to its elevated association with the ubiquitin ligase CHIP and the proteasome-associated cochaperone Bag1. Treatment of C4–12 cells with either 17-estradiol (E2) or the pure anti-estrogen ICI 182,780 (ICI) induced rapid degradation of wtER via the ubiquitin-proteasome pathway; however, in the presence of these ligands, ER-AA was less efficiently degraded. Furthermore, ER-AA was resistant to ICI-induced polyubiquitination, suggesting that these lysines are polyubiquitinated in response to the antiestrogen and demonstrate a novel role for these two lysines in the mechanism of action of ICI-induced receptor downregulation. The reduced stability of ER-AA in the unliganded state and the increased stability of ER-AA in the liganded state were concordant with reporter gene assays demonstrating that ER-AA has lower basal activity but higher E2-inducibility than wtER. These data provide the first evidence that K302/303 protect ER from basal degradation and are necessary for efficient E2 and ICI-induced turnover in breast cancer cells.