Our recent study demonstrated that aminoglycoside antibiotics are taken up into sensory hair cells of the inner ear by receptor-mediated endocytosis (E. Hashino, M. Shero, Endocytosis of aminoglycoside antibiotics in sensory hair cells, Brain Res. 704 (1995) 135–140). To elucidate the intracellular trafficking pathway of aminoglycosides following endocytotic uptake, we administered kanamycin to neonatal chicks for 1 or 5 days (400 mg/kg/day) and determined the location of kanamycin within the hair cells at various time points using immunogold electron microscopy. Quantitative and qualitative analysis of immunogold staining revealed that: (1) kanamycin was primarily localized in vesicles beneath the cuticular plate 27 h postinjection; (2) the number of vesicles per hair cell and the number of gold particles per vesicle increased over time; (3) individual vesicles tended to increase in size over time, presumably due to aggregation of smaller vesicles; and (4) in pathological hair cells, immunogold was dispersed throughout the entire subcellular region. Light microscopic observations of the basilar papilla stained with the same antibody confirmed the temporal changes in the kanamycin distribution. Moreover, results obtained from acid phosphatase cytochemistry indicated that vesicles accumulating kanamycin were mainly lysosomes. These results suggest that internalized aminoglycosides are transported via vesicular traffic into lysosomes where they accumulate over time and lead to disruption of lysosomes. The time of diffusion of kanamycin was closely related to the time of cell death, suggesting that lysosomal rupture could be a direct trigger for the hair cell degeneration.