Vitamin C and glutathione are important antioxidants that are present at millimolar concentrations in most cells and have a central role in antioxidant cellular defenses. All cells have the capacity to synthesise glutathione, while vitamin C is obtained from extracellular sources and is transported intracellularly. In vitro studies have revealed that glutathione participates in the accumulation and the maintenance of constant intracellular levels of reduced vitamin C. In vivo studies have revealed that the oxidative damage generated by glutathione deficiency is prevented by the administration of vitamin C, and the negative effects asociated with vitamin C deficiency are delayed by the administration of glutathione esters, thus indicating a two-way functional relationship between these two antioxidants. Using as models primary cultures of human umbilical vein and tonsil endothelial cells, and the endothelial cell line ECV-304, we determined the content and the role of glutathione in the cellular acumulation of vitamin C, and examined the role of vitamin C, gutathione, and the pair vitamin C-glutathione in the resistance of the endothelial cells to acute treatment with hydrogen peroxide. Although glutathione was present in the endothelial cells at similar concentrations (2-3 mM, mean ± S.E.M., n=6), the accumulation of vitamin C was glutathione independent in the tonsil endothelial cells. Acute treatment with hydrogen peroxide caused a massive decrease in intracellular glutathione, decrease which was related to the relative resistance of the cells to the treatment. Thus, cells lacking glutathion after treatment with L-buthionide(SR)sulfoximine and diethylmaleate showed a much increased sensitivity to oxidant-induced cell death, and glutathione depletion affected also the capacity of the cells to proliferate. Cells containing graded concentrations of glutathione showed a concentration-dependent increase in their resistance to oxidative stress, and a similar effect was observed in cells containing graded concentrations of ascorbic acid. However, vitamin C and glutathione were clearly different in their respective capacitie to protect cells from oxidative stress. Full protection against hydrogen peroxide treatment was observed in endothelial cells containing vitamin C concentrations as low as 0.2 mM (n=6); in contrast, at least 2 mM (n=4) glutathione was required to observe a similar protective effect. Our data are compatible with the concept that vitamin C and glutathione play a complementary role in the defense of the endothelial cells to oxidative stress, depending on the level of the oxidant insult.