In this study, we compared the uterine tissue of estrogen receptor (ER)β^−/− mice and their WT littermates for differences in morphology, proliferation [the percentage of labeled cells 2 h after BrdUrd injection and EGF receptor (EGFR) expression], and differentiation (expression of progesterone receptor, E-cadherin, and cytokeratins). In ovariectomized mice, progesterone receptor expression in the uterine epithelium was similar in WT and ERβ^−/− mice, but E-cadherin and cytokeratin 18 expression was lower in ERβ^−/− mice. The percentage of cells in S phase was 1.5% in WT mice and 8% in ERβ^−/− mice. Sixteen hours after injection of 17β-estradiol (E₂), the number of BrdUrd-labeled cells increased 20-fold in WT mice and 80-fold in ERβ^−/− mice. Although ERα was abundant in intact mice, after ovariectomy, ERα could not be detected in the luminal epithelium of either WT or ERβ^−/− mice. In both untreated and E₂-treated mice, ERα and ERβ were colocalized in the nuclei of many stromal and glandular epithelial cells. However, upon E₂ + progesterone treatment, ERα and ERβ were not coexpressed in any cells. In WT mice, EGFR was located on the membranes and in the cytoplasm of luminal epithelium, but not in the stroma. In ERβ^−/− mice, there was a marked expression of EGFR in the nuclei of epithelial and stromal cells. Upon E₂ treatment, EGFR on cell membranes was down-regulated in WT but not in ERβ^−/− mice. These findings reveal an important role for ERβ in response to E₂ and in the organization, growth, and differentiation of the uterine epithelium.