Aminoglycosides may induce irreversible hearing loss in both animals and humans. In order to study the nature and mechanisms underlying gentamicin-induced cell death in the inner ear, the cochlear neurosensory epithelia were dissected from guinea pigs and incubated with 0.5-10 mM gentamicin. Concentration-dependent loss of cell viability was detected by the inability of damaged cells to exclude propidium iodide. Outer hair cells were most sensitive towards gentamicin toxicity, followed by inner hair cells whereas Deiters and Hensen cells were not affected by the gentamicin concentrations used. The iron chelators 2,2'-dipyridyl and deferoxamine provided partial protection against gentamicin-induced hair cell death while the calcium chelator Quin-2 AM had no effect. Gentamicin (0.5-1 mM) induced condensation of chromatin typical for apoptosis. Using the fluorescent dye tetramethyl-rhodamine methyl ester and laser scanning microscopy we could visualize a loss of the mitochondrial membrane potential in damaged outer hair cells about 1 h before cell death occurred. Cyclosporin A, an inhibitor of the mitochondrial permeability pore, provided partial protection against gentamicin toxicity. This strongly suggests an involvement of the mitochondrial permeability transition in gentamicin-induced apoptosis.