The role of hormones in mammary gland development has been studied in detail using surgical and genetic models. These studies have indicated roles for estrogen in ductal elongation and terminal end bud formation. However, no comprehensive study has quantified how different doses of estrogen affect morphological parameters of mammary gland development. Additionally, comparisons between the estrogen-responsiveness of the mammary gland and uterus, the model organ for estrogen action are incomplete. In this study, immature mice were ovariectomized and implanted with osmotic pumps releasing one of eight doses of 17beta-estradiol for 10 days. As expected from the classical uterotrophic assay, the uterus showed a monotonic dose-response curve for all measured endpoints. In contrast, the mammary gland showed a non-monotonic, inverted-U shaped response to estrogen with regard to morphometric parameters, and a monotonic response with regard to gene expression parameters. These results indicate that estrogen has opposing effects in mammary gland morphogenesis depending on estrogen dose, i.e. low to moderate doses induce terminal end bud formation and ductal elongation while higher doses inhibit these processes. This non-monotonic dose-response in the mammary gland may reflect complex interactions, where estrogen can act on multiple targets either as an agonist or antagonist.