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We characterized seven factor VIII inhibitors with epitopes in the C2 domain of factor VIII using a series of factor V C2 domain chimeras that substituted exon-sized fragments of the C2 domain of factor VIII for the corresponding regions of factor V. All inhibited co-factor activity of factor VIII and six inhibited binding of factor VIII to phosphatidylserine. Inhibitors Hz, JN and GK32 bound epitopes within amino acids S2173–K2281; inhibitors GK24 and TO bound epitopes within amino acids V2223–Y2332; and inhibitors UNC11 and UNC12 bound epitopes throughout the C2 domain (amino acids S2173–Y2332). Inhibitors Hz, JN and UNC12 inhibited the co-factor activity of chimera 5A, which substituted amino acids S2173–Q2222 of factor VIII for the corresponding region of factor V, in a prothrombinase assay. This inhibition could be partially reversed by pre-incubation of chimera 5A with phospholipid vesicles, suggesting that these antibodies interfered with phospholipid binding. Inhibitors UNC11 and UNC12, on the other hand, did not inhibit the binding of chimera 1A to phosphatidylserine, suggesting that binding to the segment spanning amino acids V2282–Y2332 does not necessarily block phospholipid binding. These results agree with the model of the phospholipid-binding site determined by crystal structure of the C2 domain of factor VIII.