Free radicals are known to cause damage to biological tissues. We used histochemical methods to examine the emergence of free radicals in kanamycin (KM)-treated guinea pig cochlea. As a preliminary in vitro study, the emergence of free radicals was observed in guinea pig cochlea incubated with KM. In an in vivo study, KM was placed on the guinea pig round window membrane and the emergence of free radicals was observed over time. The emergence of free radicals was observed along the luminal membrane and hair bundles of outer hair cells. The appearance and disappearance of free radicals in the cochlea coincided with the transport of KM into the inner ear. These findings suggest that the emergence of free radicals could be attributed to the administration of KM.