Recent evidence suggests that complement plays an important role in the pathogenesis of ischaemia-reperfusion injury and that this may be mediated by reactive oxidant species (ROS). However, it is not clear how individual complement pathways could be affected by ROS. In this study purified C3 and C4 proteins were subjected to a 30 min treatment with ROS generators at 37?C: 1mM authentic peroxynitrite (ONOO-), 10 ?M/10 ?M xanthine/xanthine oxidase mixtures to generate O2- and100 ?M H2O2. We also studied the effect of ROS on complement activation in normal human serum and mannan binding lectin (MBL) deficient serums. In addition, normal human blood was taken from healthy volunteers treated with ROS during 30 min extracorporeal circulation. Sensitive ELISA assays were used to determine the effect of ROS on complement reactivity respectively and whether this related to haemolytic activities. Blood treated with the competitive ligand for MBL binding, N-acetyl-D-glucosamine (GlcNAc=100mM) and untreated blood were used as controls. There was a concentration-dependent modification of protein residues by ROS and that ROS treatment of normal and MBL deficient serums inhibited C4 deposition originating from both MBL and classical pathways and interestingly this was associated with an increased haemolytic activity. The results may suggest that ROS plays an important role in regulating complement activation independently of the classical and MBL pathways and that increased activities may be attributable to a direct effect on the alternate pathway.