Background. Factor VIII inhibitors are IgG alloantibodies that arise during replacement therapy in 25 to 50 percent of patients with severe hemophilia A. The hydrolysis of factor VIII by anti-factor VIII antibodies has been proposed as a mechanism of inactivation of factor V III. Methods. We purified IgG from patients with severe hemophilia A. The proteolytic activity of the antibodies was assessed by incubating the IgG with biotinylated human factor VIII and analyzing patterns of factor VIII cleavage by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and immunoblotting. The controls were normal human IgG and IgG purified from plasma of patients with hemophilia who did not have inhibitory antibodies. Results. Significant proteolytic activity was detected in IgG from 13 of 24 inhibitor-positive patients. No hydrolytic activity was detected in control antibodies of IgG from patients without inhibitors. The rate of hydrolysis of factor VIII by purified IgG correlated positively with the factor VIII-neutralizing activity of IgG in plasma (r 2=0.67, P=0.029). Principal-component analysis of migration profiles of digestion fragments demonstrated the heterogeneity of the catalytic potential of factor VIII inhibitors among patients. Conclusions Proteolysis is a mechanism by which IgG antibodies against factor VIII can inactivate factor VIII. Copyright © 2002 Massachusetts Medical Society.