Cell death in outer hair cells of the mammalian inner ear induced by aminoglycoside antibiotics is mediated by reactive oxygen species (ROS) and can be prevented by antioxidants. The current study investigates the role of the nuclear factor (NF)-κB pathway in cell death or survival in adult CBA mice. Kanamycin (700 mg/kg subcutaneously, twice per day) progressively destroys hair cells but after 7 days of treatment auditory function and morphology are not yet affected significantly, permitting investigations of early events in drug-induced cell death. Immunostaining for 4-hydroxynonenal, indicative of lipid peroxidation, was elevated in the cochlea, but there was no effect on nitrotyrosine, a marker for peroxynitrite. NF-κB was increased at 3 hr, 3 days, and 7 days of treatment, with p50 and p65 proteins as its most abundant subunits. Immunoreactivity for p50 was present in nuclei of inner hair cells and supporting cells that survive the drug treatment. In contrast, nuclei of outer hair cells were devoid of label. Concomitant injections of antioxidants, however, such as 2,3-dihydroxybenzoic acid or salicylate (which prevent cell death induced by kanamycin), promoted the translocation of NF-κB into the nuclei of outer hair cells. In addition, kanamycin treatment decreased tyrosine phosphorylation of the inhibitory IκBα protein, leading to increased IκBα levels in the cochlea; the effect was reversed by cotreatment with antioxidants. These results suggest that changes in the redox state of the cochlea stimulate the activation of NF-κB and that this activation is cell protective. © 2005 Wiley-Liss, Inc.