The C5b-9 monomer having the sedimentation coefficient of 23S was extracted from the rabbit erythrocyte membranes that had been treated with a limiting amount of C9-deficient human serum and of 125I-C9. Upon proteolysis by trypsin and chymotrypsin, the C9 subunits of this complex were cleaved by these enzymes at multiple sites, yielding fragments with m.w. ranging fro 40,000 to 19,000. The uncomplexed C9 was also cleaved by both enzymes at multiple sites. By contrast, the C9 subunits of the C5b-9 dimer were found to be totally insusceptible to chymotrypsin under the conditions studied (37 degrees C; 24 hr) and only partially susceptible to trypsin (33% of the C9 subunits were cleaved by trypsin into 2 fragments during incubation at 37 degrees C for up to 24 hr). Therefore, these results indicate that, although the binding of C9 molecules to the C5b-8 complex (C5b-9 monomer formation) does not significantly affect the susceptibility to proteases of the C9 molecules, C5b-9 dimer formation markedly limits the accessibility of proteases to the C9 subunit molecules. A implication of this finding to a role for C9 in C5b-9 dimerization is discussed.