An extensive plexus of nerve fibers capable of synthesizing nitric oxide was demonstrated in the cat carotid body by immunocytochemical and biochemical studies of nitric oxide synthase. Denervation experiments indicated that the axons originate from: (i) microganglial neurons located within the carotid body and along the glossopharyngeal and carotid sinus nerves, whose ramifications primarily innervate carotid body blood vessels; and (ii), sensory neurons in the petrosal ganglion, whose terminals end in association with lobules of type I cells. In the in vitro superfused cat carotid body, the nitric oxide synthase substrate, L-arginine, induced a dose-dependent inhibition of carotid sinus nerve discharge evoked by hypoxia. In contrast, the nitric oxide synthase inhibitor, L-NG-nitroarginine methylester, augmented the chemoreceptor response to hypoxia, and this effect was markedly enhanced when the preparation was both perfused and superfused in vitro. The nitric oxide donor, nitroglycerine, inhibited carotid sinus nerve discharge, and immunocytochemistry revealed that this drug stimulated the formation of cyclic 3',5'-guanosine monophosphate in both type I cells and blood vessels. Our data indicate that nitric oxide is an inhibitory neuronal messenger in the carotid body, which affects the process of chemoreceptor transduction/transmission via actions on both the receptor elements and their associated blood vessels.