Based on in vitro studies, nitric oxide (NO) is reported to be involved in initial neuronal differentiation. In order to compare this finding with the situation in vivo, we have looked for the expression of the three NO synthase isoforms in the developing mouse vestibulocochlear system. From these isoforms only the inducible NOS II is expressed during inner ear development. Examination of a series of embryonic and early postnatal animals, up to postnatal day 6, reveals a maturation-dependent, monophasic expression of this isoform. Initial expression is observed by day 10 of gestation in nerve cells of the vestibolocochlear ganglion and on their fibres. By day 14 of gestation, these afferent fibres penetrate the epithelium of the prospective receptor fields making contact with early, differentiating immunoreactive cochlear hair cells and receptor cells of the macula and crista ampullaris. This receptor-cell-derived immunoreactivity vanished in differentiated sensory hair cells by postnatal day 6, when both the constitutive isoforms and subsequent activated members of the down stream second messenger cascade (guanylate cyclase/cGMP) of the adult mouse were not then detectable. The strict phasic expression of NOS-II, independent of the second messenger system mentioned above, implies that there is a unique role for the inducible NOS isoform in nerve cell differentiation, independent of the NO/guanylate cyclase/cGMP pathway.