Using cultured human endothelial cells, we investigated the contribution of concentrations of magnesium to the antioxidant system and oxidative stress. Cells were cultured at decreasing magnesium levels (569, 380, 190 and 95 microM) for 72 h. We then measured the amount of released hydrogen peroxide (H2O2) from the cells, the consumption of exogenous H2O2, the intracellular reduced glutathione (GSH) and the oxidized glutathione (GSSG) contents and the activities of glutathione reductase and catalase. Magnesium at a level of 949 microM was used as a control. The effect of magnesium deficiency on cellular membrane permeability was determined by measurement of the amount of [14C] amino acid mixture released from the cells. The results showed that during 72 h of magnesium-deficient treatment, the H2O2 release from the cells gradually increased and consumption of exogenous H2O2 was enhanced during the first 48 h of treatment. GSH content gradually decreased but GSSG was not affected. The activity of glutathione reductase was first stimulated and then inhibited. Catalase activity was gradually reduced. [14C]Amino acid mixture release from the cells continuously increased. We suggest that magnesium deficiency affected the intracellular antioxidant system in cultured endothelial cells.