Non-adrenergic non-cholinergic nerves provide the main inhibitory autonomic supply to intestinal smooth muscle and other organ systems. Nitric oxide is likely to act as a neurotransmitter in these nerves and a nitric oxide synthase has been demonstrated in autonomic neurons. However, there are as yet no biochemical measurements of nerve-induced release of nitric oxide or its breakdown products nitrite and nitrate. We have examined the possibility that nitric oxide is released by stimulation of autonomic nerves in the guinea-pig intestine by studying the release of nitric oxide, nitrite and nitrate. The biological activity of a vascular relaxing factor released by the activation of these nerves was compared with that of nitric oxide using a bioassay system as previously described. Nitrite and nitrate release were measured by high-performance liquid chromatography using UV absorbance. The relaxation of the bioassay tissues to nerve stimulation was indistinguishable from the relaxation induced by nitric oxide. Both relaxations were equally unstable and inhibited to a similar degree by haemoglobin and enhanced by superoxide dismutase. Furthermore, the release of the relaxing factor was attenuated by treatment with the nitric oxide synthase inhibitor N omega-nitro-L-arginine. Concomitant with the release of the relaxing factor, which was frequency dependent, there was a frequency-dependent release of nitrite and nitrate in amounts sufficient to explain the vascular relaxations observed during nerve stimulation. The release of nitrite and nitrate was also inhibited by treatment with the nitric oxide synthase inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)