Over the past two decades, considerable progress has been made in understanding the mechanisms underlying aminoglycoside ototoxicity. Aminoglycoside damage progresses from cochlear base to apex and from outer to inner hair cells. Aminoglycoside antibiotics enter hair cells at the apical pole and are taken up into lysosomes and mitochondria. Aminoglycosides generate damaging reactive oxygen species that can initiate a program of cell death. In cases of acute ototoxicity involving high doses of aminoglycosides, hair cells appear to die by apoptosis and their cuticular plates can be ejected from epithelium. However, after long-term systemic administration, hair cell death occurs by either necrosis or apoptosis. Aminoglycoside ototoxicity is exacerbated by loop diuretics that accelerate drug entry into cochlear fluids resulting in rapid cell death. Knowledge of the signaling pathways involved in aminoglycoside ototoxicity has led to new strategies for preventing ototoxicity, involving antioxidants, protease inhibitors and neurotrophic factors. Such fundamental knowledge may one day lead to clinical procedures that block the ototoxic side effects of this important class of antibiotics.