The structural elements of the NO-cGMP signaling pathway have been described in the vestibular peripheral system. However, the functions of NO in the vestibular endorgans are still not clear. We evaluated the action of NO on the Ca²⁺ currents in hair cells isolated from the semicircular canal crista ampullaris of the rat (P14 -18) by using the whole- and perforated-cell patch-clamp technique. The NO donors 3-morpholinosydnonimine (SIN-1), sodium nitroprusside (SNP), and (±)-(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide (NOR-4) inhibited the Ca²⁺ current in hair cells in a voltage-independent manner. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) prevented the inhibitory effect of SNP on the Ca²⁺ current. The selective inhibitor of the soluble form of the enzyme guanylate cyclase (sGC), 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), also decreased the SNP-induced inhibition of the Ca²⁺ current. The membrane-permeant cGMP-analogue 8-Br-cGMP mimicked the SNP effect. KT-5823, a specific inhibitor of cGMP-dependent protein kinase (PGK), prevented the inhibition of the Ca²⁺ current by SNP and 8-Br-cGMP. In the presence of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent that prevents the S-nitrosylation reaction, the SNP effect on the Ca²⁺ current was significantly diminished. These results demonstrated that NO inhibits in a voltage-independent manner the voltage-activated Ca²⁺ current in rat vestibular hair cells via the activation of a cGMP-signaling pathway and through a direct action on the channel protein via a S-nitrosylation reaction. The inhibition of the Ca²⁺ current by NO may contribute to the regulation of the intracellular Ca²⁺ concentration and hair-cell synaptic transmission.