Proteolytic activation of human Factor VIII (VIIIa) by thrombin was correlated with the generation of a light-chain-derived 73(71) kDa polypeptide plus polypeptides of 51 and 43 kDa derived from the heavy chain(s). Factor VIIIa activity was unstable and decayed to an inactive form (VIIIi) in the absence of additional proteolysis. The subunit structure of Factor VIIIa was studied using two rapid chromatographic methods. Gel filtration of Factor VIIIa showed that coagulant activity was correlated with the 73 and 51 kDa polypeptides which co-eluted with a Stokes radius of 46 A and was separated from the 43 kDa fragment. A similar polypeptide elution pattern was obtained for Factor VIIIi following prolonged incubation with thrombin. Gel filtration of EDTA-inactivated Factor VIIIa showed that the 73 and 51 kDa polypeptides eluted separately with Stokes radii of 32 and 38 A, respectively. Anion-exchange HPLC of Factor VIIIa resolved the coagulant-active 73/51 kDa dimer from the inactive dimer. The labile activity of Factor VIIIa was stabilized by chemical crosslinking reagents, presumably by formation of intra-chain crosslinks. A native Mr of 136,000 for Factor VIIIa, calculated from its Stokes radius (46 A) and sedimentation coefficient (7.1 S), was compatible with a non-covalent dimer composed of 73 and 51 kDa subunits.