OBJECTIVE/HYPOTHESIS: Mammalian auditory hair cells that are unable to regenerate and various agents, including gentamicin, can irreversibly damage the hair cells. Erythropoietin, known as the primary regulator of erythropoiesis, exerts also neuroprotective effects by binding to its receptor. We tested whether erythropoietin can protect the hair cells from gentamicin-induced damage. STUDY DESIGN: This study localized the erythropoietin receptor in the cochlea and analyzed the effect of erythropoietin on gentamicin-damaged hair cells in vitro. METHODS: Expression of erythropoietin receptor in the rat cochlea was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Protection of auditory hair cells from gentamicin was tested in vitro by exposing cultured rat organs of Corti with increasing concentrations of erythropoietin (0.1 U/mL, 1 U/mL, and 10 U/mL). RESULTS: We detected erythropoietin and erythropoietin receptor mRNA expression in the organ of Corti, spiral ganglion, and stria vascularis by RT-PCR. Immunohistochemistry revealed that the erythropoietin receptor localizes to the outer and inner hair cells and supporting cells of the organ of Corti, as well as to the spiral ganglion cells and the stria vascularis. Significantly less hair cell loss occurred in the organs of Corti that were pretreated with 0.1 U/mL erythropoietin as compared with samples treated with gentamicin only. CONCLUSION: Decreased hair cell loss in erythropoietin-treated organs of Corti that had been exposed to gentamicin provides evidence for a protective effect of erythropoietin in aminoglycoside-induced hair cell death.