Estradiol 17-β (E₂) induces epithelial proliferation, stratification, and cornification in vaginal epithelium. Our aim was to determine the respective roles of epithelial and stromal estrogen receptor-α (ERα) in these E₂-induced events. Vaginal epithelium (E) and stroma (S) from adult ERα knockout (ko) and wild-type (wt) neonatal Balb/c mice were enzymatically separated and used to produce four types of tissue recombinants in which epithelium, stroma, or both lack functional ERα. Tissue recombinants were grafted into female nude mice, which were subsequently ovariectomized and treated with oil or E₂. In response to E₂ treatment, grafts prepared with wt-S (wt-S + wt-E and wt-S + ko-E) showed similar large increases in epithelial labeling index, indicating that E₂ stimulated epithelial proliferation despite a lack of epithelial ERα in wt-S + ko-E tissue recombinants. Conversely, in tissue recombinants prepared with ko-S (ko-S + wt-E and ko-S + ko-E), epithelial labeling index remained at baseline levels after E₂ or oil treatment, even though epithelial ERα were detected in ko-S + wt-E grafts. Epithelial cornification was present in wt-S + wt-E grafts from E₂-treated hosts, whereas epithelium in all other tissue recombinants failed to cornify. Grafts composed of wt-S + wt-E from E₂-treated hosts had highly stratified epithelium, whereas epithelial thickness was reduced almost 60% in wt-S + ko-E tissue recombinants grown in E₂-treated hosts and was atrophic in all other tissue recombinants. In addition, cytokeratin 10, a marker of epithelial differentiation, was strongly expressed in wt-S + wt-E tissue recombinants grown in E₂-treated hosts but was markedly reduced or absent in all other tissue recombinants. These results indicate that E₂-induced vaginal epithelial proliferation is mediated indirectly through stromal ERα, consistent with our recent findings in uterus. Conversely, both epithelial and stromal ERα are required for E₂-induced cornification and normal epithelial stratification. These are the first known functions attributed to epithelial ERα in vivo and the first time any epithelial response to E₂ has been shown to involve both stromal and epithelial ERα.