The two human umbilical vein endothelial cell-derived lines, ECRF24 and ECV304, differ in responsiveness to oxidative stress. In confluent monolayers of ECRF24, but not in ECV304, peroxides induce stress responses such as plasma membrane blebbing and nuclear condensation. The peroxide effect on ECRF24 was preceded by oxidation of reduced glutathione (GSH) and of NAD(P)H, and by oxidation of the redox-sensitive probe, chloromethyl 2',7'-dichlorofluorescin (DCFH). In monolayers of ECV304, peroxides induced only minimal oxidation of GSH, NAD(P)H and DCFH, which was associated with a greatly reduced GSH peroxidase activity in these cells. However, in spite of the absence of a blebbing response, ECV304 were more susceptible than ECRF24 to membrane lipid peroxidation and peroxide-induced necrosis. Only for ECV304, the culturing with high levels of polyunsaturated fatty acids increased lipid peroxidation and cellular death. Treatment of these cells with the GSH peroxidase mimic ebselen effectively reversed their decreased vitality. We conclude that, in peroxide-treated endothelial cells, cell death (necrosis) can result from lipid peroxidation by peroxide that has not been removed by GSH peroxidases, whereas extensive peroxidase activity may cause a stress response (blebbing). The data further identify ECV304 as a stress-sensitive cell line, where peroxides exert their effects independently of GSH oxidation.