The anticomplementary factor of cobra venom (CoVF) was found to activate the terminal complement (C) components (C5 through C9) in mouse or rat serum or plasma in the presence of EDTA. The same concentrations of EDTA would block C activation by EA, zymosan, and inulin and would block C activation in human and guinea pig serum by all of these activators, including CoVF. Murine serum was found to contain two factors that allowed CoVF-induced C activation and bystander hemolysis in the presence of EDTA. The first was factor B or a related component of the alternative pathway, which allowed formation of a functionally active complex of CoVF and factor B in the presence of EDTA. The second was murine C5, which led to augmented hemolysis of unsensitized bystander erythrocytes and activation of the other terminal components. Preformed complexes of CoVF and rat factor B were active in leading to C5 and C6 consumption in human or guinea pig serum that had been fortified with rat C5. In contrast to native CoVF, these complexes did not lead to electrophoretic conversion of C3 in the human or guinea pig serum. It was further shown that in the presence of rat C5, purified CoVF led to moderate activation of the terminal components in human serum-EDTA in the absence of electrophoretic conversion of C3, indicating that in these situations, CoVF activated C5 and the other terminal components with little or no utilization of C3.