Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the pre-pubertal gland, and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution and then increased again post-weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double labeling experiments in animals treated with E+P for three days demonstrated that Stat5a was localized exclusively to cells containing both estrogen and progesterone receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland, and suggest that these hormones act at the cellular level through their cognate receptors.