Reactive oxygen species (ROS) have been postulated to be involved in drug ototoxicity and noise-induced hearing loss. Hydrogen peroxide (H(2)O(2))-induced cell damage in the inner ear was investigated using the neurosensory epithelium of a guinea pig cochlea. Hair cells and supporting cells of the epithelium incubated in Hanks' balanced salt solution were viable up to 6 h. After 2 h of treatment with 0.2 mM H(2)O(2) about 85% of the outer hair cells lost their viability. In contrast inner hair cells slowly began to die after 2 h of H(2)O(2) treatment. The Deiters cells and Hensen cells did not show any signs of damage in the presence of H(2)O(2). Nifedipine, a calcium channel blocker, Quin-2 AM, an intracellular calcium chelator, and 2,2'-dipyridyl, a membrane-permeable iron chelator, all provided partial protection against H(2)O(2)-induced outer hair cell death. The combination of both chelators showed an additional protective effect. The antioxidants N-acetylcysteine and glutathione-monoethyl ester completely protected against H(2)O(2) damage. These results suggest that calcium, iron, and thiol homeostasis play a crucial role in hair cell death caused by H(2)O(2).