We investigated the binding of C4 and C3 to red cell surfaces by non-complement enzymes. Cell bound C components were quantitated by a radioimmunoassay, the chain structure of bound components was analyzed by Western blotting and the hemolytic activity of bound components was determined. Trypsin, chymotrypsin, plasmin, elastase, thrombin, kallikrein and enzymes from Bacillus subtilis, Staphylococcus aureus and Streptomyces griseus all were found capable of binding C4b and C3b to sheep red cells. C4b bound by any of these enzymes was hemolytically active; both classical and alternate pathway activity of C3 could be demonstrated for most enzymes except plasmin and thrombin. In addition, trypsin and the bacterial enzymes were also able to generate the classical pathway C3-convertase from C4b + C2. The hemolytic efficiency of enzyme bound C4b and C3b was about the same as for these molecules bound by complement enzymes. In contrast, the process of binding by the non-complement enzymes was several hundred-fold less efficient than by cell bound complement enzymes. The results demonstrate that several enzymes can replace the C1 and C42 enzymes in the classical pathway and are able to initiate the alternative pathway by activating C3 and binding C3b to the cell surface.