Mammary glands from the estrogen receptor-α knockout (αERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted ERα signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute to the αERKO mammary phenotype. We report that circulating PRL is reduced in the female αERKO mouse. Implantation of an age-matched, heterozygous ERα pituitary isograft under the renal capsule of 25-day-old or 12-week-old αERKO mice increased circulating PRL and progesterone levels, and induced mammary gland development. Graftedα ERKO mice also possessed hypertrophied corpora lutea demonstrating that PRL is luteotropic in the αERKO ovary. By contrast, ovariectomy at the time of pituitary grafting prevented mammary gland development in αERKO mice despite elevated PRL levels. Hormone replacement using pellet implants demonstrated that pharmacological doses of estradiol induced limited mammary ductal elongation, and estradiol in combination with progesterone stimulated lobuloalveolar development. PRL alone or in combination with progesterone or estradiol did not induce αERKO mammary growth. Estradiol and progesterone are required for the structural development of the αERKO mammary gland, and PRL contributes to this development by inducing ovarian progesterone levels. Therefore, the manifestation of the αERKO mammary phenotype appears due to the lack of direct estrogen action at the mammary gland and an indirect contributory role of estrogen signaling at the hypothalamic/pituitary axis.