Estrogen induces proliferation of uterine epithelium through a paracrine action of estrogen receptor (ERα) in the underlying stroma. In ovariectomized mice primed with progesterone, estrogen stimulates proliferation in both the epithelium and the stroma. We set out to test whether a paracrine mode of action is involved in estrogen-induced proliferation of the uterine stroma. Epithelial and mesenchymal tissues derived from uteri of neonatal ERα null mice (ERαKO) or wild-type mice were separated and recombined in all four possible configurations (ERα or ERα⁻ epithelium with ERα or ERα⁻ mesenchyme) and grafted into female athymic mice. After 5 wk, hosts were ovariectomized and challenged with hormone treatment, and cellular proliferation was monitored by thymidine autoradiography. Results showed that, although the full response of the epithelium was dependent on an ERα-positive mesenchyme, stromal cell proliferation was independent of tissue ERα. This latter observation suggests that the response of the stroma was due to a systemic factor induced in the ERα-positive hosts. To test this possibility, pieces of whole uterus from neonatal wild-type or ERαKO mice were grafted into syngeneic wild-type or ERαKO hosts. In these whole-uterus grafts, estradiol stimulated ERαKO uterine stroma when they were grown in wild-type hosts but not when grown in ERαKO hosts. The epithelium of whole-uterus ERαKO grafts did not respond to estrogen, regardless of the host phenotype. These observations suggest that treatment of progesterone-primed mice with estradiol stimulates production of a systemic factor that is capable of inducing uterine stromal cell proliferation and that this systemic factor is produced by an ERα-dependent mechanism.