Recently, a family of growth factors has been described that activates erbB-2 receptors. These factors, known as the neu differentiation factors (NDF) or heregulins (HRG), induce tyrosine phosphorylation of erbB-2 receptors as a result of their direct interaction with either erbB-3 or erbB-4 receptors. Although it is known that expression of erbB-2 receptors has relevance in human breast cancer progression, how erbB-2, -3 and -4 receptors regulate mammary epithelial cell proliferation is not known. Therefore, experiments were carried out to study the mitogenic activity of NDF/HRG on the human mammary epithelial cell line MCF-1 OA which can be cultured continuously under serum-free conditions. MCF-1 OA cells, like primary cultures of normal human mammary epithelial cells, express an absolute requirement for exogenous epidermal growth factor (EGF) and insulinlike growth factor I (IGF-I) for growth. The results of these experiments indicate that NDF/HRG can induce tyrosine phosphorylation of pl85erbB-2 in MCF-1OA cells and is mitogenic for these cells. This is consistent with the coexpression of erbB-2 and erbB-3 mRNA that we have observed in MCF-1 OA cells. In addition, we found that NDF/HRG can substitute for either EGF or IGF-I to stimulate proliferation of these cells. The ability to substitute for both EGF and IGF-I is a unique property of NDF/HRG and is not shared by other members of the EGF or IGF family of growth factors, nor by other factors that we have studied. A striking isoform specificity was also observed which indicated that the p-isoforms of NDF/HRG were greater than ten times more mitogenic than the a-isoforms. We also examined the mitogenic activity of NDF/HRG on MCF-1OA cells that overexpress the erbB-2 receptor as a result of infection with a retroviral vector containing the human c-erbB-2 gene (MCF-1 OAerbB-2 cells). These studies indicated that MCF-1 OAerbB-2 cells have increased sensitivity to the mitogenic effects of NDF/ HRG and that these cells are responsive to the a-isoforms of NDF/HRG at physiological concentrations. Thus, NDF/HRG is a dual specificity growth factor for human mammary epithelial cells, and the responsiveness of the cells to NDF/HRG is influenced by the level of expression of erbB-2 receptors.