ABSTRACT: Hearing loss induced by ototoxicity is a worldwide problem despite the development of newer antibiotics and chemotherapy agents. The cellular mechanisms responsible for aminoglycoside-induced hearing loss are still poorly understood. We have developed two different methods of studying the dynamic cellular and subcellular changes in the chick auditory sensory epithelium that occur during hair cell death. The first study was performed in mature chicks after a single, high dose injection of gentamicin, which results in the rapid loss of all hair cells in the basal third of the cochlea. Chicks were sacrificed at discrete time points after drug treatment, and transmission electron microscopy was performed to study the ultrastructural changes in basal hair cells during the course of cell death. We noted various changes in the cell morphology including accumulation of cytoplasmic inclusion bodies, dispersion of the cytoplasmic polyribosomes, mitochondrial swelling, and cellular extrusion by 24 h after injection. The next two studies were performed using tissue cultures from mature avian auditory sensory epithelium. Cultured cells were labeled using vital fluorescent markers, and levels of intracellular calcium and reactive oxygen species within hair cells were studied following aminoglycoside exposure. We identified a dose-dependent increase in the levels of intracellular calcium, which was blocked by an inhibitor of voltage-gated calcium channels. We also found that levels of reactive oxygen species in hair cells greatly increased after exposure to gentamicin, and this response was blocked by two different antioxidants. These studies serve to identify key cellular and molecular changes in hair cells in response to ototoxic drugs. Further study of these processes may lead to a better understanding of how ototoxicity is induced and to potential preventative interventions.