Nitric oxide (NO)-mediated neurotoxicity may be an appropriate pathophysiological model with which to explain a variety of inner ear diseases characterized by acute or progressive hearing loss, tinnitus and vertigo. The localization of NO synthase (NOS) isoforms was examined in the inner ear of the pigmented guinea pig after intratympanic injection of 1 mg lipopolysaccharide (LPS) or 5 mg gentamicin (GM) using an immunohistochemical method, revealing the expression of NOS II in the inner ear. Production of NO in the isolated organ of Corti and utricle or in the isolated vestibular and cochlear hair cells after stimulation with L-arginine, glutamate, GM and LPS was investigated using the fluorescence indicator 4,5-diaminofluorescein diacetate. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, glutamate, GM and LPS. A significant increase in NO production was also noted in the LPS-treated animals. These findings imply that NO from constitutive NOS may mediate ototoxicity in the early phase, whereas NO from NOS II may contribute to the late phase of tissue damage in the inner ear. Based on this hypothesis, reduction of glutamatergic excitotoxicity and inhibition of NOS, scavenging superoxide and scavenging peroxynitrite are thought to attenuate NO-mediated otoneurotoxicity.