Netilmicin, a semisynthetic aminoglycoside antibiotic, is less ototoxic in a variety of species than other aminoglycosides currently in therapeutic use. In this study, mixed-breed cats (four/group) were given daily sc injections of netilmicin (20, 40, and 80 mg/kg), gentamicin (20 and 40 mg/kg), or tobramycin (20, 40, and 80 mg/kg) for up to 30 weeks or until ototoxicity was observed. The animals were examined throughout the study for effects on cochlear and vestibular function. Hematologic, serum chemical, and drug-serum (24-hr postdose) assays were performed at approximate monthly intervals during the dosing period. The cochleae, kidneys, and liver were examined microscopically. The mean number of dose days required to produce vestibulotoxic effects, demonstrated by impaired righting reflex or locomotor ataxia, was from 41 to 61 in cats dosed with tobramycin (40 and 80 mg/kg) or gentamicin. No vestibular dysfunction was observed in any of the netilmicin 20-mg/kg-dosed cats, in two cats each of the tobramycin 20-mg/kg and netilmicin 40-mg/kg groups, and in one netilmicin 80-mg/kg-dosed animal. Histologic examination of the cochleae revealed degeneration of the hair cells and supporting sensory structures in the majority of cats dosed with gentamicin at 20 and 40 mg/kg and tobramycin at 40 and 80 mg/kg. Less than 50% of the tissues from cats of the tobramycin 20-mg/kg and netilmicin 40- and 80-mg/kg-dosed groups had similar degenerative cochlear changes. No cochlear damage was noted in any of the cats given netilmicin at 20 mg/kg. Results of the clinical laboratory determinations were generally unremarkable. Proximal tubular degeneration was the principal finding observed in the kidneys of the animals. Under the conditions of this study, at least a twofold (vestibular) to fourfold (cochlear) relative safety margin for ototoxicity was established in favor of netilmicin over tobramycin and gentamicin.