Immuno-gold electron microscopy was used to assess the uptake pathways of aminoglycoside antibiotic kanamycin (KM) in sensory hair cells. Accumulation of gold particles was evident on the plasma membrane as well as in large smooth vesicles beneath the apical surfaces of hair cells 12 h after a systemic administration of KM. Immuno-gold was exclusively localized in the vesicles 27 h post-injection. Cationic ferritin, a membrane-bound insoluble marker, was colocalized with KM in the vesicle structures after their simultaneous in vitro application. These results strongly suggest that KM is taken up into sensory hair cells via receptor-mediated endocytosis at their apical surfaces. In addition, the profound time lag between KM uptake and hair cell death suggests involvement of targeting mechanisms in cytotoxic signalling pathways of the drugs.