The purpose of this study was to investigate the occurrence of free radicals, nitric oxide (NO), superoxide (O^–₂) and peroxynitrite, in the inner ear of the guinea pig following intratympanic injection with 5 mg of gentamicin (GM). Forty-eight hours after GM injection, varying degrees of degeneration of the inner ear were observed. Immunohistochemical study revealed immunoreactivity to NO synthase II (which generates NO) and to xanthine oxidase (which generates O^–₂) in both the vestibular organ and the organ of Corti. Immunohistochemical investigation, using a specific antinitrotyrosine antibody, also showed intense staining, suggesting formation of peroxynitrite in the inner ear through the reaction of NO with O^–₂. Scanning electron-microscopic study showed that the ototoxic effects could be blocked with N-nitro-L-arginine methylester, a competitive inhibitor of NO synthase, with superoxide dismutase, an O^–₂ scavenger, and with ebselen, a scavenger of peroxynitrite. On the basis of these findings, it can be concluded that NO together with O^–₂, which form more reactive peroxynitrite, play an important role in GM ototoxicity in the guinea pig.