Mammary glands from the estrogen receptor-a knockout (aERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted ERa signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hor- mones that contribute to the aERKO mammary phenotype. We report that circulating PRL is reduced in the female aERKO mouse. Im- plantation of an age-matched, heterozygous ERa pituitary isograft under the renal capsule of 25-day-old or 12-week-old aERKO mice increased circulating PRL and progesterone levels, and induced mam- mary gland development. Grafted aERKO mice also possessed hy- pertrophied corpora lutea demonstrating that PRL is luteotropic in the aERKO ovary. By contrast, ovariectomy at the time of pituitary grafting prevented mammary gland development in aERKO mice despite elevated PRL levels. Hormone replacement using pellet im- plants 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 aERKO mammary growth. Estradiol and progesterone are required for the structural development of the aERKO mammary gland, and PRL contributes to this development by inducing ovarian progester- one levels. Therefore, the manifestation of the aERKO mammary phenotype appears due to the lack of direct estrogen action at the mammary gland and an indirect contributory role of estrogen sig- naling at the hypothalamic/pituitary axis. (Endocrinology 141: 2982- 2994, 2000)