Although it is known that, in the uterus, estrogen receptor α (ERα) is involved in proliferation and progesterone receptor in differentiation, the role of the two other gonadal-hormone receptors expressed in the uterus, androgen receptor (AR) and estrogen receptor β (ERβ), remains undefined. In this study, the involvement of AR in 17β-estradiol (E₂)-induced cellular proliferation in the immature rat uterus was investigated. AR levels were low in the untreated immature uterus, but 24 h after treatment of rats with E₂, there was an increase in the levels of AR and of two androgen-regulated genes, IGF-I and Crisp (cysteine-rich secretory protein). As expected, E₂ induced proliferation of luminal epithelial cells. These actions of E₂ were all blocked by both the antiestrogen tamoxifen and the antiandrogen flutamide. The E₂-induced AR was found by immunohistochemistry to be localized exclusively in the stroma, mainly in the myometrium, where it colocalized with ERα but not with ERβ. ERβ, detected with two different ERβ-specific antibodies, was expressed in both stromal and epithelial cells either alone or together with ERα. Treatment with E₂ caused down-regulation of ERα and ERβ in the epithelium. The data suggest that, in E₂-induced epithelial cell proliferation, ERα induces stromal AR and AR amplifies the ERα signal by induction of IGF-I. Because AR is never expressed in cells with ERβ, it is unlikely that ERβ signaling is involved in this pathway. These results indicate an important role for AR in proliferation of the uterus, where estrogen and androgen do not represent separate pathways but are sequential steps in one pathway.