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 1 A 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.