Therapeutic ototoxic drugs are one of the major causes of damage in the peripheral auditory system, leading to hearing loss. In this study, we have examined the toxic actions of three classes of ototoxins (sodium salicylate, gentamicin and cisplatin) in organotypic cultures of postnatal cochlear explants. In these cultures, afferent innervation of hair cells by primary auditory neurons remained intact. Double labelling with a monoclonal antibody against neurofilament protein and a phalloidin-fluorescein isothiocyanate conjugate revealed that the three types of drugs induced differential damage to auditory neurons and hair cells in the cochlea. While gentamicin preferentially caused hair cell death, sodium salicylate specifically induced degeneration of auditory neurons. In contrast, cisplatin resulted in destruction of both auditory neurons and hair cells. Neuronal degeneration was largely prevented by the addition of neurotrophin-4/5, brain-derived neurotrophic factor and neurotrophin-3 to the culture media together with the ototoxins, while nerve growth factor and other growth factors had no effect. In contrast, the hair cell loss caused by cisplatin or gentamicin was not attenuated by the presence of neurotrophins. These results suggest that ototoxic mechanisms of salicylates, aminoglycosides and chemotherapeutic agents are different. Auditory neuronal loss induced by ototoxins may be prevented by specific neurotrophins.