Vascular dysfunction is a hallmark of many diseases, including coronary heart disease, stroke, and diabetes. The underlying mechanisms of these disorders are intimately associated with an increase in oxidative stress and excess generation of reactive oxygen species (ROS). Here we report that the anionic free radical, superoxide (O₂^.-), directly affects the function of ion channels in vascular endothelial cells. Vascular endothelial cells were exposed to O₂^.- under physiological, symmetrical chloride and chloride free conditions. Superoxide was generated from the reaction of xanthine (0.2 mM) and xanthine oxidase (0.1-, 1- and 10 mU/ml) while its effects were determined with the whole-cell mode of the patch-clamp technique. Inhibitors of K⁺, and Cl^- channels were used to determine the role of these ion channels in mediating the electrophysiological effects of superoxide. Addition of O₂^.- caused a dose-dependent depolarization of endothelial cells and activation of the whole-cell current. Activation of superoxide-dependent current was observed in the presence of inhibitors of K⁺ channels, Ba²⁺ (100 µM) or iberiotoxin (100 nM), and was not affected by inhibitors of non-selective cation channels, La³⁺, or by inhibition of the Cl^-/HCO₃^- transporter by bumetanide. The inhibitors of the Cl^- channel, NPPB (0.1 mM) or DIDS (100 µM), partially prevented activation of superoxide-dependent current but were unable to reverse it. The effects of superoxide on the amplitude of whole-cell current were prevented and reversed by superoxide dismutase. Taken together, these results suggest that superoxide directly affects the function of ion channels in vascular endothelium but the mechanisms of its modulatory effects remain unresolved.