Estrogen receptor-alpha (ER) is down-regulated in the presence of its cognate ligand, estradiol (E2), as well as in the presence of antiestrogens, through the ubiquitin proteasome pathway. Here, we show that, at pharmacological concentrations, the degradation rate of pure antagonist/endogenous ER complexes from human breast cancer MCF-7 cells is 10 times faster than that of ER-E2 complexes, while 4-hydroxy-tamoxifen (4-OH-T)-ER complexes are stable. Whereas pure antagonist-ER complexes are firmly bound to a nuclear compartment from which they are not extractable, the 4-OH-T-ER accumulates in a soluble cell compartment. No difference was observed in the fate of ER whether bound to pure antiestrogens ICI 182,780 or RU 58668. Cycloheximide experiments showed that, while the proteasome-mediated destruction of E2-ER (unlike that of RU 58668- and ICI 182,780-ER) complexes could implicate (or not) a protein synthesis-dependent process, both MAPKs (p38 and ERKs p44 and p42) are activated. By using a panel of kinase inhibitors/activators to study the impact of phosphorylation pathways on ER degradation, we found that protein kinase C is an enhancer of proteasome-mediated degradation of both ligand-free and ER bound to either E2, 4-OH-T, and pure antagonists. On the contrary, protein kinase A, MAPKs, and phosphatidyl-inositol-3 kinase all impede proteasome-mediated destruction of ligand free and E2-bound ER while only MAPKs inhibit the degradation of pure antiestrogens/ER species. In addition, no correlation was found between the capacity of kinase inhibitors to affect ER stability and the basal or E2-induced transcription. These results suggest that, in MCF-7 breast cancer cells, ER turnover, localization, and activity are maintained by an equilibrium between various phosphorylation pathways, which are differently modulated by ER ligands and protein kinases.