Complement plays an important role in ischemia-reperfusion injury. We recently demonstrated that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) activated the classical complement pathway and augmented iC3b deposition. In the present study, we investigated the potential role of oxygen-derived free radicals, NF-kappaB and new protein synthesis in this model. HUVECs subjected to 12 or 24 h hypoxic stress (1% O2) and then reoxygenated (0.5, 1, 2 or 3 h; 21% O2) in 30% human serum activated complement and deposited iC3b. Addition of hydrogen peroxide (H2O2; 1-100 micromol/l) to normoxic HUVECs increased iC3b deposition in a concentration-dependent manner. H2O2 (10 micromol/l), a concentration that did not significantly increase iC3b deposition on normoxic HUVECs, augmented iC3b deposition on hypoxic/reoxygenated HUVECs. We observed a significant increase in intracellular H2O2 and hydroxyl radical (OH.) production in hypoxic/reoxygenated HUVECs using dihydrorhodamine 123. Further, treatment of HUVECs with dimethylthiourea (DMTU, 1-100 micromol/l), deferoxamine (DEF, 1-100 micromol/l), or oxypurinol (10 micromol/l), but not superoxide dismutase (SOD, 500 U/ml), catalase (300 U/ml) or iron-loaded DEF, attenuated iC3b deposition following hypoxia/reoxygenation in a concentration-dependent manner. Western analysis demonstrated hypoxia-induced nuclear NF-kappaB translocation that increased with reoxygenation. Inhibition of new protein synthesis (i.e. cycloheximide) or inhibition of NF-kappaB (ALLN or SN-50) also significantly decreased iC3b deposition on hypoxic/reoxygenated HUVECs. We conclude that (1) hypoxic/reoxygenated HUVECs generate H2O2 and OH.; (2) treatment of HUVECs with cell permeable reactive oxygen species inhibitors/scavengers (i.e. DEF, DMTU, oxypurinol) but not large molecular weight inhibitors (i.e. catalase or SOD) significantly reduces iC3b deposition and (3) inhibition of new protein synthesis or NF-kappaB activation attenuates iC3b deposition. These data suggest that iC3b deposition on the vascular endothelium may be regulated by intracellular oxygen-derived free radical-induced activation of NF-kappaB, new protein synthesis and activation of the classical complement pathway during ischemia/reperfusion.