In combination with androgens, estrogens can induce aberrant growth and malignancy of the prostate gland. Estrogen action is mediated through two receptor subtypes: estrogen receptors α (ERα) and β (ERβ). Wild-type (wt) and transgenic mice lacking a functional ERα (αERKO) or ERβ (βERKO) were treated with the synthetic estrogen diethylstilbestrol (DES). DES induced prostatic squamous metaplasia (SQM) in wt and βERKO but not in αERKO mice, indicating an essential role for ERα, but not ERβ, in the induction of SQM of prostatic epithelium. In order to determine the respective roles of epithelial and stromal ERα in this response, the following tissue recombinants were constructed with prostatic epithelia (E) and stroma (S) from wt and ERKO mice: wt-S+wt-E, αERKO-S+αERKO-E, wt-S+αERKO-E, and αERKO-S+wt-E. A metaplastic response to DES was observed in wt-S+wt-E tissue recombinants. This response to DES involved multilayering of basal epithelial cells, expression of cytokeratin 10, and up-regulation of the progesterone receptor. Tissue recombinants containing αERKO-E and/or -S (αERKO-S+αERKO-E, wt-S+αERKO-E, and αERKO-S+wt-E) failed to respond to DES. Therefore, full and uniform epithelial SQM requires ERα in the epithelium and stroma. These results provide a novel insight into the cell–cell interactions mediating estrogen action in the prostate via ERα.