Glutathione (GSH) plays a key role in cellular antioxidant defenses by scavenging reactive oxygen species and reducing lipid peroxides. Intracellular GSH levels are regulated by transport of its precursor L-cystine via system xc-, which can be induced by oxidant stress. As oxidatively modified low density lipoproteins (LDLs) contribute to impaired vascular reactivity and the formation of atherosclerotic lesions, we have examined the effects of oxidized LDL and the antioxidant vitamins C and E on the L-cystine-GSH pathway in human umbilical artery smooth muscle cells (HUASMCs). Oxidized LDL, but not native LDL, elevated intracellular GSH levels and L-cystine transport via system xc- in a time-dependent (up to 24 hours) and dose-dependent (10 to 100 microg x mL-1) manner. These increases were dependent on protein synthesis and the extent of LDL oxidation, but the induction of L-cystine transport activity was independent of GSH synthesis. Pretreatment of HUASMCs for 24 hours with vitamin E (100 micromol/L) attenuated oxidized LDL-mediated increases in GSH, whereas pretreatment with vitamin C depressed basal levels and abolished oxidized LDL-induced increases in GSH and L-cystine transport in a time-dependent (3 to 24 hours) and dose-dependent (10 to 100 micromol/L) manner. Pretreatment of cells with dehydroascorbate had no effect on oxidized LDL-mediated increases in L-cystine transport and only marginally attenuated increases in GSH. Our findings provide the first evidence that vitamin C spares endogenous adaptive antioxidant responses in human vascular smooth muscle cells exposed to atherogenic oxidized LDL.