We recently described the isolation from human serum of a serum protein (C4 binding protein) that functions as an essential cofactor for C3b inactivator in the proteolysis of fluid-phase C4b and to a much lesser extent, C3b. We show here the role of C4 binding protein in the formation and function of the classical pathway C3 convertase (C42). C4 binding protein interferes with the assembly of the membrane-bound C3 convertase of the classical pathway and accelerates the decay of C42 in a dose-dependent fashion. Its removal from serum by means of specific immune absorption promotes the vigorous consumption of C3 after addition of C1; this effect is abolished by reconstitution with purified C4 binding protein. Although C4 binding protein inhibits the hemolytic function of cell-bound C4b, we did not detect any change in the structure of C4b even after prolonged incubations of EAC14 with C4 binding protein. For this reason, and on the basis of studies of the time required for maximal reactivity (Tmax) of cellular intermediates generated in the presence of C4 binding protein and limited amounts of C2, we conclude that the effects of C4 binding protein are probably mediated by displacing C2a from specific binding sites on C4b. In addition, C4 binding protein enhances the cleavage by C3b inactivator of the alpha' chain of cell-bound C4b. When EAC14 cells were incubated with both control proteins, the Tmax of the cells was prolonged and the lysis was markedly diminished. We conclude that C4 binding protein and C3b inactivator control the C3 convertase of the classical pathway in a fashion similar to that described for beta 1H and C3b inactivator in the alternative pathway.