High glucose (HG) concentrations are toxic to various cells in vivo, but cells become insensitive to HG toxicity when they are subcultured serially in vitro. Oxidative stress is involved in HG toxicity, and metal ions, especially iron, mediate some oxidative stress. To investigate mechanisms involved in the insensitiveness of cultured cells to HG toxicity, we focused on the level of intracellular iron. Freshly prepared human umbilical vein endothelial cells contained a substantial amount of iron, whereas its level decreased rapidly during the course of culture (to less than 10%). The iron content was restored by incubation of the cells with Fe(III)/8-hydroxyquinoline, and the iron-supplemented cells were more susceptible to both oxidant- and HG-induced injury. Under the HG conditions, the iron-loaded cells were subjected to higher levels of oxidative stress. The enhanced HG toxicity by iron was attenuated by the treatment with several antioxidants including catalase, ascorbic acid, and pyruvate. These data suggested that the insensitiveness of subcultured cells to HG toxicity is, at least in part, due to rapid and dramatic loss of intracellular iron. Supplementation with iron is useful to restore the vulnerability of cultured cells to HG that is normally observed in in vivo situations.