Free radicals are suggested to play an important role in hypoxic-ischemic neuronal death. However, the importance in human disease is not known. Furthermore, whether posthypoxic free radical formation mainly occurs in endothelium and neutrophils, or whether neuronal production is important, is not finally determined. To study this we differentiated human Ntera2 teratocarcinoma cells into postmitotic NT2-N neurons and exposed them to free radicals, hypoxia, or oxygen and glucose deprivation. These cells are devoid of nitric oxide synthase, and we hypothesized that free radicals are important mediators downstream of N-methyl-D-aspartate stimulation. Production of free radicals, evaluated with the fluorescent dyes dihydrorhodamine and 2',7'-dichlorodihydrofluorescein, was significantly higher in neurons deprived of oxygen and glucose after 40 min of reoxygenation than in normoxic cells. The antioxidant trolox, the flavonoid quercetin, thiopental, and the N-methyl-D-aspartate-glutamate receptor antagonist MK-801 reduced the formation of free radicals. Treatment with the flavonoid rutin (86 +/- 16% of hypoxic cells without drug, p < 0.01), trolox (86 +/- 20%, p < 0.01), and MK-801 (57 +/- 12%, p < 0.01) reduced lactate dehydrogenase release after 6 h of hypoxia. Trolox, salicylate, and quercetin also significantly reduced lactate dehydrogenase release after 3 h of oxygen and glucose deprivation. The protection offered by these antioxidants was, however, limited compared with the effect of MK-801. We conclude that oxygen and glucose deprivation causes a moderate increase in the formation of free radicals in NT2-N neurons that can be inhibited by antioxidants and by blocking of the N-methyl-D-aspartate-glutamate receptor. Although MK-801 conveys profound protection, antioxidants provide only a limited improvement in neuronal survival. Thus in this model, mechanisms downstream of the N-methyl-D-aspartate-glutamate receptor other than free radicals and nitric oxide have to be invoked.