Epidermal growth factor receptor (EGFR) levels were assayed by an enzyme-linked immunosorbent assay (ELISA) in microdissected organ of Corti (OC) from neonatal rats directly after isolation and after 3 days in culture with and without neomycin treatment. In addition, the cellular distribution of the EGFR in the OC was determined by immunohistochemistry. The in vitro level of EGFR determined by ELISA assays doubled after neomycin damage to OC, suggesting that EGFR is subject to up-regulation following this treatment. Immunohistochemistry of both in vivo and in vitro controls indicates that EGFR is predominantly localized in the stereociliary bundles of the hair cells; supporting cells and the apical junctions between the remaining Kolliker organ cells were also immunolabeled. In neomycin-treated cultures, sensory cells were degenerated, so no labeling could be seen. However, supporting and Kolliker organ cells continued to show labeling. In addition, nerve fibers in the region of the future osseous spiral lamina and projecting out toward the damaged sensory epithelium were immunostained. The up-regulation of the EGFR and its redistribution within the OC following neomycin damage support the earlier observation that growth factors that act through EGFR, such as EGF and transforming growth factor-alpha can induce neonatal mammalian auditory hair cell replacement under culture conditions, after aminoglycoside treatment.