Oxidative stress acutely increases the permeability of the vascular endothelium to large molecules that would not otherwise cross the barrier. Ascorbic acid is an antioxidant that tightens the endothelial permeability barrier, so we tested whether it might also prevent the increase in endothelial permeability due to cellular oxidative stress. Treatment of EA.hy926 endothelial cells cultured on filter inserts with H(2) O(2) , menadione, and buthionine sulfoximine increased endothelial permeability to radiolabeled inulin. Short-term ascorbate loading of the cells to what are likely physiologic concentrations of the vitamin by treating them with dehydroascorbate prevented the increase in endothelial permeability due to these agents. The nonphysiologic antioxidants dithiothreitol and tempol also prevented increases in endothelial barrier permeability induced by the agents. These results suggest that oxidative stress induced directly by oxidants or indirectly by glutathione depletion impairs endothelial barrier function and that intracellular ascorbate may serve to prevent this effect.