Damage to auditory hair cells in the inner ear as a consequence of aging, disease, acoustic trauma, or exposure to ototoxins underlies most cases of hearing impairment. Because the mammalian ear cannot replace damaged hair cells, loss of hearing is irreversible and progressive throughout life. One of the current goals of inner ear biology is to develop therapeutic strategies to prevent hair cell degeneration. Although important progress has been made in discovering factors that mediate hair cell death, very little is known about the molecular pathway(s) that signal survival. Here we considered the role of NF-kappaB, a ubiquitous transcription factor that plays a major role in the regulation of many apoptosis- and stress-related genes, in mediating hair cell survival. NF-kappaB was detected in a constitutively active form in the organ of Corti of 5-day-old rats. Selective inhibition of NF-kappaB through use of a cell-permeable inhibitory peptide in vitro caused massive degeneration of hair cells within 24 h of inhibitor application. Hair cell death occurred through an apoptotic pathway through activation of caspase-3 and may involve transcriptional down-regulation of the gadd45beta gene, an anti-apoptotic NF-kappaB target. In view of our results, it seems likely that NF-kappaB may participate in normal hair cell function.