Estrogen plays important roles in preparing mammary tissue for lactation. However, estrogen also influences innervation in some tissues. We examined the effect of estrogen on peripheral innervation of mammary tissues of ovariectomized adult virgin female rats. Seven days after ovariectomy, 17beta-estradiol or placebo pellets were implanted subcutaneously, and tissues were harvested 1 week later. Estrogen treatment decreased mammary gland mass and adipocyte content, while ductal content increased and vascular composition was unaffected. Estrogen increased total areas occupied by nerves in mammary gland sections immunostained for the pan-neuronal marker protein gene product 9.5, and this increase persisted after normalizing for treatment-induced differences in gland mass. Although a significant increase in tyrosine hydroxylase-immunoreactive sympathetic nerve area was observed, no difference was detected following correction for differences in gland size, implying a conserved number of sympathetic nerves in the face of reduced gland volume. Calcitonin gene-related peptide-immunoreactive sensory nerve sectional area was also increased, and corrected nerve area remained 88% greater, indicating nerve proliferation during estrogen treatment. Total, sensory, and sympathetic innervation of the nipple and adjacent dermal tissue were unaffected by estrogen. We conclude that chronic estrogen elevation induces selective proliferation of rat mammary gland calcitonin gene-related peptide-containing nerves, which are associated primarily with blood vessels and are probably nociceptors. Because they are likely to subserve a vasodilatory function, increased innervation may promote increased blood flow necessary for milk formation during suckling. Moreover, these findings may help explain abundant anecdotal reports of increased breast sensitivity in humans under high estrogen conditions.