OBJECTIVE To examine the inhibition by L-cysteine (Cys) and L-homocysteine (HoCys) of NO-induced relaxation of aorta. METHODS The tension of rabbit aortic rings in oxygenated Krebs' solution was recorded isometrically. RESULTS Pretreatment of endothelium-denuded rings with Cys or HoCys inhibited the NO-induced increase in cGMP. The inhibitory effects of Cys or HoCys on relaxation responses to subsequent additions of NO 75 nmol.L-1 gradually diminished with time, which was consistent with the loss of the sulfhydryl concentration of Cys and HoCys. Superoxide dismutase (SOD) 35 kU.L-1 attenuated the inhibition by Cys and HoCys of NO-induced relaxation. Neither boiled SOD nor catalase 100 kU.L-1 antagonized the inhibitory effects of Cys. Preaddition of SOD 35 kU.L-1 inhibited the reduction of cytochrome C by Cys. Increasing concentrations of SOD from 35 to 350 kU.L-1 intensified the cytochrome C reduction. Addition of xanthine 300 mumol.L-1 plus xanthine oxidase 1 U.L-1 to the mixture of cytochrome C 60 mumol.L-1 and Cys 100 mumol.L-1 produced an additional augmentation of SOD-inhibitable reduction of cytochrome C. The rate of the reduction of cytochrome C induced by HoCys 100 mumol.L-1 was much slower than with Cys. Addition of NO reduced the SH concentrations of both the supernatant of aortic homogenate and Cys in Krebs' solution. CONCLUSIONS The inhibition by the SH compounds of NO is mediated partly by the superoxide generated by the auto-oxidation of these compounds, and partly by a direct reaction of SH groups with NO.