1. This study examined the possibility that an S-nitrosothiol, rather than nitric oxide, functions as the non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitter in the bovine retractor penis (BRP) muscle. 2. Treatment of BRP muscle with either of two sulphydryl inactivating agents, diamide (1 mM) and N-ethylmaleimide (0.3 mM), inhibited NANC relaxation and this was prevented by pretreating tissues with L-cysteine (3 mM), L-glutathione (3 mM) or dithiothreitol (3 mM). Inhibition was not specific, however, since the inactivating agents also inhibited the relaxant actions of authentic nitric oxide (0.3 microM), glyceryl trinitrate (0.001-1 microM) and isoprenaline (0.01-1 microM). 3. Reacting nitric oxide with L-cysteine in nominally oxygen-free solution at pH 3, followed by purging to remove free nitric oxide and neutralisation, produced greater and more prolonged relaxant activity when assayed on rabbit aortic rings than could be attributed to nitric oxide alone. H.p.l.c. analysis of the mixture identified a new peak distinct from either L-cysteine or nitric oxide which was responsible for the relaxant activity. The spectral absorption of this new compound had two bands with peaks at 218 and 335 nm. 4. Using a series of structural analogues of L-cysteine (all at 15 mM) it was found that removal of the carboxyl group (L-cysteamine), replacement of the carboxyl with an ester function (L-cysteine methyl ester) or substitution at the amino group (N-acetyl-L-cysteine) had no effect on the ability to generate relaxant activity upon reaction with nitric oxide (0.1 mM). In contrast, substitution at the sulphydryl group (S-methyl-L-cysteine, L-cysteinesulfinic acid and L-cysteic acid), or formation of disulphides(L-cystine and L-cystamine) led to a complete loss of ability to generate relaxant activity. L-Glutathione was also able to react with nitric oxide to produce relaxant activity, and this too was blocked upon substitution of the free sulphydryl group (S-methyl-L-glutathione). A free sulphydryl group was therefore required to generate relaxant activity following reaction with nitric oxide.5. Reacting L-cysteine (10 mM) with nitric oxide (~ 3 mM) under more stringent oxygen-free conditions followed by purging to remove free nitric oxide resulted in the generation of low relaxant activity and small absorption peaks at 218 and 335 nm and these were unaffected upon exposure to the air. In contrast, admitting air to the reaction chamber before purging enhanced both relaxant activity and the absorption peaks at 218 and 335 nm by some 40 fold and the solution turned pink due to the appearance of another absorption peak at 543 nm. This enhanced relaxant activity was not due to nitrogen dioxide being the reactive species, since at 0.1 mM this gas failed to react with L-cysteine to generate relaxant activity, and at 1 mM generated less activity than the equivalent concentration of nitricoxide.6. The relaxant activity generated by reacting nitric oxide with L-cysteine or L-glutathione was abolished following treatment with haemoglobin (3 MicroM), methylene blue (10 MicroM) or Nmethylhydroxylamine(100 MicroM), but was unaffected by N0-nitro-L-arginine (30 MicroM). Furthermore, two agents that generate superoxide anion, pyrogallol (0.1 mM) and hydroquinone (0.1 mM), also inhibited this relaxant activity as well as that induced by authentic nitric oxide (0.3 MicroM) but as previously reported, had no effect on relaxation induced by NANC nerve stimulation. Superoxide dismutase(100 u ml1) reversed the actions of pyrogallol and hydroquinone but had no effect on NANC relaxation.7. In conclusion, the reaction of nitric oxide with L-cysteine or L-glutathione generates relaxant activity which exceeds that of nitric oxide alone and probably results from formation of S-nitrosocysteine and S-nitrosoglutathione, respectively. The effects of pyrogallol and hydroquinone suggest that the NANCneurotransmitter is a superoxide anion-resistant, nitric oxide-releasing molecule and that neither S-nitrocysteine nor S-nitrosoglutathione is a suitable candidate for this.