The effect of magnesium (Mg)-deficient culture on endothelial cell susceptibility to oxidative stress was examined. Bovine endothelial cells were cultured in either control sufficient (0.8 mM) or deficient (0.4 mM) levels of MgCl2. Oxygen radicals were produced extracellularly by the addition of dihydroxyfumarate and Fe(3+)-ADP. Isolated Mg-deficient endothelial cells produced 2- to 3-fold higher levels of thiobarbituric acid (TBA)-reactive materials when incubated with this free radical system. Additional studies were performed using digitized video microscopy and 2',7'-dichlorofluorescein diacetate (DCFDA) as an intracellular indicator for oxidative events at the single cell level. In response to the exogenous oxidative stress, endothelial cells exhibited a time-dependent increase in fluorescence, suggestive of intracellular lipid peroxidation. The increase in cellular fluorescence began within 1 min of free radical addition; the Mg-deficient cells exhibited a more rapid increase in fluorescence than that of Mg-sufficient cells. In separate experiments, cellular viability was assessed using the Trypan blue exclusion assay. Mg deficiency increased cytotoxicity of the added oxyradicals, but the loss of cellular viability began to occur only after 15 min of free radical exposure, lagging behind the detection of intracellular oxidation products. These results suggest that increased oxidative endothelial cell injury may contribute to vascular injury during Mg deficiency.