Exposure to estrogen in the neonatal period affects prostatic growth and leads to an increased incidence of prostatic intraepithelial neoplasia in later life. The effects of neonatal estrogen are clearly dependent on estrogen receptor (ER) alpha because they do not occur in ERalpha-knockout mice. Because ERalpha is expressed in the stroma, but not in the epithelium, of the adult ventral prostate, the concept of indirect estrogen effects through stromal signaling has been proposed. Here, we show that during the first 4 weeks of life, there are profound and rapid changes in the ER profile in the mouse ventral prostate. ERalpha is abundant in the stroma during week 1, but by week 2 it is exclusively epithelial, and then by week 4, ERalpha is lost and ERbeta is dominant in the prostatic epithelium. The presence of ERalpha is associated with a high proliferation index, and ERbeta is associated with quiescence. Branching morphogenesis was altered in ERalpha-/-, but not in ERbeta-/-, mice. We conclude that imprinting and branching morphogenesis of the ventral prostate are mediated by estrogen acting directly on epithelial and stromal ERalpha during the first 2 weeks of life.