The in vivo interaction of estrogen receptor (ER) and Hsp90, demonstrated in the absence of hormone by a nuclear cotranslocation assay of the cytoplasmic Hsp90 with the karyophilic receptor, was disrupted by agonist and antagonist ligands, which, after dissociating the Hsp90, allowed the chaperone protein to be relocalized in the cytoplasm. The pure antiestrogen RU 58668 (RU), which was unable to stimulate an estrogen-dependent reporter gene and completely inhibited its estradiol-induced activity, also profoundly modified the subcellular distribution of ER in a specific time- and dose-dependent manner; ER appeared as speckled fluorescent clusters mainly located in the perinuclear region of the cytoplasm. The kinetics of appearance and reversal of the RU-dependent ER mislocalization in the presence or absence of cycloheximide demonstrated 1) that this effect was reversed by RU withdrawal or estradiol (E2) treatment, and 2) that cycloheximide with RU inhibited and reversed the ER cytoplasmic mislocalization induced by RU alone. These results point to a protein synthesis-dependent step in the mechanism of action of this antiestrogen. After RU treatment, a large portion of ER was found in the particulate fraction of the cytoplasm. However, confocal and electron microscopic analysis showed that ER clusters were not associated with specific cytoplasmic organelles or compartments. Using ER mutants, it was found that the ligand binding domain was sufficient for RU to produce receptor mislocalization, while the constitutive nuclear localization signals were dispensable. We propose that the antiestrogenic properties of RU are primarily due to the induction of an aggregation-prone receptor conformation that cannot undertake the constitutive and the ligand-induced nuclear localization function of the receptor because it is sequestered in the cytoplasm by fast turning over protein(s). We predict that antiestrogens able to block ER nuclear localization will behave as pure antihormones and will inhibit all the nuclear action of ER elicited by agonistic ligands or by ligand-independent mechanisms such as growth factor stimulation.