Aminoglycoside antibiotics are ototoxic, inducing irreversible sensorineural hearing loss mediated by oxidative and excitotoxic stresses. The NF-kappaB pathway is involved in the response to aminoglycoside damage in the cochlea. However, the molecular mechanisms of this ototoxicity remain unclear. We investigated the expression of PKCzeta, a key regulator of NF-kappaB activation, in response to aminoglycoside treatment. Amikacin induced PKCzeta cleavage and nuclear translocation. These events were concomitant with chromatin condensation and paralleled the decrease in NF-kappaB (p65) levels in the nucleus. Amikacin also induced the nuclear translocation of apoptotic inducing factor (AIF). Prior treatment with aspirin prevented PKCzeta cleavage and nuclear translocation. Thus, aspirin counteracts the early effects of amikacin, thereby protecting hair cells and spiral ganglion neurons. These results demonstrate that PKCzeta acts as sentinel connecting specific survival pathways to mediate cellular responses to amikacin ototoxicity.