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Apoptosis may play an important role in the mechanism of aminoglycoside ototoxicity. Caspases and calpains are regarded to be important factors in the regulation of cell death in the inner ear. We hypothesized that caspase or calpain inhibitors would protect hair cells from aminoglycoside ototoxicity. In order to test this hypothesis we carried out a pilot study to determine if gentamicin (GM) would induce caspase and calpain immunolabeling in guinea pig hair cells. Having confirmed this we carried out the main experiment using guinea pig vestibular organ culture to determine if caspase and calpain would protect the hair cells from GM ototoxicity. Immunoreactivity for caspase-3 and m-calpain was detected in the vestibular sensory cells and ganglia after GM treatment. Both caspase and calpain inhibitors protected hair cells against gentamicin ototoxicity. It is suggested that inhibition of apoptosis is essential in order to block aminoglycoside ototoxicity.