—Hyperhomocysteinemia is associated with endothelial dysfunction, although its mechanism is unknown. Isometric tension recordings and lucigenin chemiluminescence were used to assess the effects of homocysteine exposure on endothelium-dependent and -independent relaxation in isolated rabbit aortic rings and superoxide anion (O₂⁻) production by cultured porcine aortic endothelial cells, respectively. Homocysteine (0.1 to 10 mmol/L) produced a significant (P<0.001) concentration- and time-dependent inhibition of endothelium-dependent relaxation in response to both acetylcholine and the calcium ionophore A23187. Only the intracellular O₂⁻ scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron, 10 mmol/L) significantly (P<0.001) inhibited the effect of homocysteine on acetylcholine- and A23187-induced relaxation. Incubation of porcine aortic endothelial cells with homocysteine (0.03 to 1 mmol/L for up to 72 hours) caused a significant (P<0.001) time-dependent increase in the O₂⁻ released by these cells on the addition of Triton X-100 (1% [vol/vol]), with levels returning to values comparable to those of control cells at the 72-hour time point. These changes in O₂⁻ levels were associated with a time-dependent increase in endothelial cell superoxide dismutase activity, becoming significant (P<0.001) after 72 hours. Furthermore, the homocysteine-induced increase in endothelial cell O₂⁻ levels was completely inhibited (P<0.001) by the concomitant incubation with either Tiron (10 mmol/L), vitamin C (10 μmol/L), or vitamin E (10 μmol/L). These data suggest that the inhibitory effect of homocysteine on endothelium-dependent relaxation is due to an increase in the endothelial cell intracellular levels of O₂⁻ and provide a possible mechanism for the endothelial dysfunction associated with hyperhomocysteinemia.