The present study was designed to test the hypothesis that Hcys reduces intracellular nitric oxide concentrations ([NO]_i) and stimulates superoxide (O₂^.-) production in the renal arterial endothelium, thereby resulting in endothelial dysfunction. Using fluorescence microscopic imaging analysis, a calcium ionophore, A23187 (2 µM) and bradykinin (2 µM) were found to increase endothelial [NO]_i in freshly-dissected lumen-opened small renal arteries loaded with DAF-2DA (10 µM). Pre-incubation of the arteries with L-Hcys (20-40 µM) significantly attenuated the increase in endothelial [NO]_i. However, L-Hcys had no effect on nitric oxide synthase (NOS) activity in the renal arteries, as measured by a conversion rate of 3H-arginine to ³H-citrulline, but it concentration-dependently decreased DAF-2-sensitive fluorescence induced by PAPA NONOate in the solution, suggesting that L-Hcys reduces endothelial [NO]_i by its scavenging action. Since other thiol compounds such as L-cysteine and glutathione were also found to reduce [NO]_i, it seems that decreased NO is not the only mechanism resulting in endothelial dysfunction or arteriosclerosis in hHcys. By analysis of intracellular O₂^.- levels using dihydroethidium trapping, we found that only L-Hcys among the thiol compounds studied markedly increased O₂^.- levels in the renal endothelium. These results indicate that L-Hcys inhibits agonist-induced NO increase, but stimulates O₂^.- production within endeothelial cells. These effects of L-Hcys on [NO]_i and [O₂^.-] may contribute to endothelial injury associated with hHcys.