Most evidence supports the view that ER alpha is responsible for estrogen (ovarian estradiol, E(2))-induced proliferation in the epithelial cells of the mammary gland, but despite this, proliferating epithelial cells do not express ER alpha. We have examined this apparent paradox by studying the role of ER alpha and ER beta in E(2)-induced proliferation in mammary glands (measured by BrdUrd incorporation into DNA) in mice with intact ER beta (WT mice) and those in which the ER beta gene has been inactivated (ER beta(-/-) mice). On treatment of ER beta(-/-) mice with E(2) or ovariectomized WT mice with E(2), tamoxifen, or a specific ER beta agonist (BAG), the number of BrdUrd-labeled cells in mammary glands increased from 3.4% in controls to 28-38% in the treated mice. This indicates that both ER alpha and ER beta can mediate E(2)-induced proliferation independently of each other. With specific antibodies, ER beta was found in both epithelial and stromal cells, whereas ER alpha was strictly epithelial. Within 4 h of a single dose of E(2), ER alpha was lost from the nuclei of epithelial cells. In WT mice, ER alpha reappeared by 24 h, but in ER beta(-/-) mice, return to the nucleus was delayed by 24 h. At 4 h after E(2), neither ER alpha nor progesterone receptor was detectable in BrdUrd-labeled nuclei but by 48 h after E(2), 29% of the BrdUrd-labeled cells expressed ER alpha, and 21-38% expressed progesterone receptor. During 3 weeks of continuous E(2) treatment, ER beta remained in the nucleus, but there was no detectable ER alpha. With tamoxifen treatment, ER alpha remained in the nucleus, but ER beta was lost. From these results, we conclude that ER alpha receives the proliferation signal from E(2), initiates DNA synthesis, and is then lost from cells. The subsequent steps in proliferation can proceed in the absence of either ER alpha or ER beta. ER beta facilitates the return of ER alpha to the nucleus and restores responsiveness to E(2). By down-regulating ER beta, tamoxifen may prolong refractoriness to E(2) in mammary epithelium.