Investigations into the mechanism of alternative pathway-dependent lysis of C4b-coated cells are reported. Test cells (EAC1q4b) were formed by reaction of sheep erythrocytes with antibody, C1 and C4. In C5-deficient serum, more C3b was deposited onto EAC1qC4b than onto control cells (EAC1q). The possibility that the C4bBb enzyme could form was considered, but no C3 convertase activity was generated when magnesium, properdin and factors B and D were added to EAC1qC4b. Binding studies employing radiolabeled components provided evidence that C4b bound the C3 convertase, C3bBbP, through a weak interaction with C3b. These data implied C3 conversion would be localized to the cell surface, thereby amplifying C3b deposition. This could be demonstrated in vitro. C3b, properdin, factor B and factor D were all required and the amplified C3b deposition was not due to deposition onto C4b itself. In serum, C5 convertase activity would be consequently expressed and cell lysis would result. This could be the mechanism by which the sera of C2-deficient patients mediate lysis of antibody coated sheep erythrocytes.