Hair cell loss due to acoustic and ototoxic damage often leads to hearing and balance impairments. Although a spontaneous event in chicks and lower vertebrates, hair cell replacement occurs at a much lower frequency in mammals presumably due to a very low rate of supporting cell proliferation following injury. We report here that heregulin, a member of the neuregulin family, dramatically enhances proliferation of supporting cells in postnatal rat utricular epithelial sheet cultures after gentamicin treatment, as revealed by bromo-deoxyuridine (BrdU) immunocytochemistry. A dose-dependent study shows that the maximal effects of heregulin are achieved at 3 nM. The mitogenic effects of heregulin are confirmed in utricular whole mount cultures. Autoradiography of the utricular whole mount cultures shows that heregulin also enhances the number of tritiated thymidine-labeled cells within the hair cell layer. TaqMan quantitative RT-PCR analysis and immunocytochemistry reveal that heregulin and its binding receptors (ErbB-2, ErbB-3 and ErbB-4) are expressed in the inner ear sensory epithelium. Of several ligands activating various ErbB receptors, including heregulin, neuregulin-3, beta-cellulin, heparin binding-epidermal growth factor (HB-EGF), transforming growth factor-alpha (TGF-alpha) and EGF, heregulin shows the most potent mitogenic effects on supporting cells. Because neuregulin-3 that signals only through ErbB-4 does not show an effect, these data suggest that activation of the ErbB-2-ErbB-3 heterodimeric complexes, rather than ErbB-4, is critical for the proliferative response in the utricular sensory epithelium. In addition, gentamicin treatment induces an upregulation of heregulin mRNA. Considered together, heregulin may play an important role in hair cell regeneration following ototoxic damage.