Endothelial barrier dysfunction after exposure to fatty acids may be mediated by disturbances in antioxidant protection. To evaluate this hypothesis, components of the glutathione redox cycle such as total, reduced, and oxidized glutathione and glutathione reductase and peroxidase were measured in cultured porcine endothelial cells exposed to 90 mumol/L of stearic acid (18:0), oleic acid (18:1 omega-9), linoleic acid (18:2 omega-6), linolenic acid (18:3 omega-3), and/or buthionine sulfoximine (BSO). Treatment with fatty acids caused an initial decrease in glutathione concentrations, which was followed by an increase at later time points. The most marked changes in glutathione redox cycle components were induced by 18:2. BSO increased susceptibility of fatty acid-mediated injury to endothelial monolayers. The results indicate a fundamental role of the glutathione redox cycle in protection against fatty acid-mediated injury to the vascular endothelium.