We recently demonstrated that extracellular adenosine 5′-triphosphate (ATP) induced nitric oxide (NO) production in the inner hair cells (IHCs) of the guinea pig cochlea, which inhibited the ATP-induced increase in the intracellular Ca2+ concentrations ([Ca2+]i) by a feedback mechanism [Shen, J., Harada, N. & Yamashita, T. (2003) Neurosci. Lett., 337, 135–138]. We herein investigated the role of the NO–cGMP pathway and neuronal NO synthase (nNOS) in the ATP-induced Ca2+ signalling in IHCs using the Ca2+-sensitive dye fura-2 and the NO-sensitive dye DAF-2. Fura-2 fluorescence-quenching experiments with Mn2+ showed that ATP triggered a Mn2+ influx. L-NG-nitroarginine methyl ester (L-NAME), a nonspecific NOS inhibitor, accelerated the ATP-induced Mn2+ influx while S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, suppressed it. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase, and KT5823, an inhibitor of cGMP-dependent protein kinase, enhanced the ATP-induced [Ca2+]i increase. 8-Bromoguanosine-cGMP, a membrane-permeant analogue of cGMP mimicked the effects of SNAP. Moreover, the effects of 7-nitroindazole, a selective nNOS inhibitor, mimicked the effects of L-NAME regarding both the enhancement of the ATP-induced Ca2+ response and the attenuation of NO production. Immunofluorescent staining of nNOS using a single IHC revealed that nNOS was distributed throughout the IHCs, but enriched in the apical region of the IHCs as shown by intense staining. In conclusion, the ATP-induced Ca2+ influx may be the principal source for nNOS activity, which may interact with P2X receptors in the apical region of IHCs. Thereafter, NO can be produced and conversely inhibits the Ca2+ influx via the NO–cGMP–PKG pathway by a feedback mechanism.