Epidermal growth factor (EGF) is a multifunctional regulator of mammary epithelial cells (MEC) that transduces its signals through the EGF receptor (EGFR). To clarify the role of the EGFR in the mammary gland, EGFR expression, localization and function were examined during different developmental stages in rats. Immunoblot analysis demonstrated high levels of EGFR during puberty, pregnancy and involution as well as at sexual maturity, and low levels throughout lactation. An immunohistochemical assay was used to show that EGFR was distinctly expressed in a variety of cell types throughout mammary glands from virgin rats and rats during pregnancy and involution, and was down-regulated in all cell types throughout lactation. To examine the relationship between EGFR expression and function, primary MEC were cultured under conditions that induced physiologically relevant growth, morphogenesis and lactogenesis. Cultured MEC expressed an in vivo-like profile of EGFR. EGFR was high in immature MEC, down-regulated in functionally differentiated MEC, and then up-regulated in terminally differentiated and apoptotic MEC. An inhibitor of the tyrosine kinase domain of EGFR was used to demonstrate that EGFR signaling was required for growth and differentiation of immature MEC, and for survival of terminally differentiated MEC, but not for maintaining functional differentiation.