Complete mammary gland development takes place following puberty and depends on the estrogen receptor (ER)alpha and the progesterone receptor (PR) and is tightly regulated by the interaction of the mammary epithelium with the stromal compartment. Studies using mammary tissues of immature mice have indicated that stromal but not epithelial ER alpha is required for mammary gland growth. This study investigates whether these same tissue growth requirements of neonate tissue are necessary for mammary development and response in adult mice. Mammary epithelial cells were isolated from adult mice with a targeted disruption of the ER alpha gene (alpha ERKO) or from wild-type counterparts and injected into epithelial-free mammary fat pads of 3-wk-old female alpha ERKO or wild-type mice. Ten weeks after cell injection, analysis of mammary gland whole mounts showed that both stromal and epithelial ER alpha were required for complete mammary gland development in adult mice. However, when the mice were treated with high doses of estradiol (E2) and progesterone, stromal ER alpha was sufficient to generate full mammary gland growth. Surprisingly, ER alpha-deficient epithelial cells were able to proliferate and develop into a rudimentary mammary ductal structure in an ER alpha-negative stroma, indicating that neither stromal nor epithelial ER alpha are required for the mammary rudiment to form in the adult mouse, as confirmed by the phenotype of the alpha ERKO mammary gland. Use of this in vivo model system has demonstrated that neonatal and adult mammary tissues use a different tissue-specific role for ER alpha in mammary response. Immunostaining for ER alpha and PR in the mammary outgrowths supported the view that both stromal and epithelial ER alpha, in cooperation with epithelial PR, govern mammary gland development in adult mice.