The relationship between end-stage renal disease (ESRD), hemodialysis, and oxidative stress is controversial. To determine whether ESRD causes oxidative stress, we measured basal levels of plasma F2-isoprostanes as a marker of lipid peroxidation in vivo, and peroxynitrite-stimulated formation of F2-isoprostanes, as a marker of the oxidizibility of plasma lipids in vitro, before and after routine hemodialysis.Total plasma F2-isoprostanes were measured by gas chromatography-mass spectrometry (GC-MS) before and after the oxidation of plasma lipids with the peroxynitrite-generating compound, 3-morpholino-sydnonimine (SIN-1), in 23 patients with ESRD patients undergoing regular hemodialysis, and 14 controls. Plasma vitamin E concentrations were measured by high-performance liquid chromatography (HPLC).There was no difference in basal plasma concentrations of F2-isoprostanes in the ESRD group prior to hemodialysis, 246 +/- 20 pg/mL, compared to controls, 252 +/- 28 pg/mL, or immediately on completion of hemodialysis, 236 +/- 14 pg/mL. Incubation of control plasma with SIN-1 caused the formation of F2-isoprostanes with plasma concentrations increasing to 987 +/- 54 pg/mL at 6 hours. The formation of F2-isoprostanes stimulated by SIN-1 was markedly enhanced in the plasma obtained from patients undergoing hemodialysis at 1861 +/- 174 pg/mL, P < 0.001, and SIN-1-induced formation of F2-isoprostanes was further increased in plasma obtained immediately after hemodialysis at 2437 +/- 168 pg/mL, P < 0.001. Incubation of plasma with SIN-1 resulted in the net consumption of vitamin E.Although basal plasma F2-isoprostanes were similar in patients with ESRD compared with controls, the presence of oxidative stress in patients with ESRD was unmasked when the plasma was stressed by peroxynitrite generated from SIN-1, and this was enhanced further by hemodialysis.