Based on organ bath experiments illustrating nitric oxide (NO) or an NO-releasing substance as mediator of the nonadrenergic noncholinergic (NANC) nerve-induced relaxations in the canine ileocolonic junction and rat gastric fundus, a bioassay superfusion technique was developed to detect and characterize the inhibitory NANC neurotransmitter. Evidence is provided that NANC nerve stimulation results in the release of a vasorelaxant factor with pharmacological properties similar to NO: its release is blocked by inhibition of the NO biosynthesis and tetrodotoxin, but enhanced by L-arginine. Its half-life is comparable to that of NO, and its biological activity is enhanced by superoxide dismutase, but abolished by hemoglobin. In addition, the nitrergic transferable factor is similarly affected as authentic NO by pyrogallol, hydroquione, hydroxocobalamin and L-cysteine. Nitrosothiols, like S-nitroso-L-cysteine, S-nitrosoglutathione and S-nitroso-N-acetyl-D,L- penicillamine, on the other hand, have a different pharmacological profile compared to NO and the nitrergic factor, indicating that NO, and not a nitrosothiol, is released from inhibitory NANC nerves in the canine ileocolonic junction. This nerve-induced release is Ca(2+)-dependent and prejunctionally regulated by K+ channels and alpha 2-adrenoceptors: blockade of K+ channels enhances the release, whereas alpha 2-adrenoceptor activation reduces the release of the nitrergic factor, possibly by activating K+ channels.