Factor VIII (F. VIII) in normal plasma: a) shortens the prolonged clotting time of hemophilic plasma (F. VIII coagulant, VIII:C), b) precipitates with heterologous antisera (F. VIII related antigen, VIIIR:AG), and c) together with the antibiotic Ristocetin, aggregates platelets (F. VIII related Willebrand factor, VIIIR:WF). VIII:C has been shown by others to be separable from VIIIR:AG-WF proteins by gel filtration with buffers of high ionic strength. No one has, to our knowledge, clearly separated VIIIR:WF from VIIIR:AG. We seem to have accomplished this by sequential use of two antibodies to F. VIII. The IgG fractions of a precipitating rabbit anti-human VIII and of a human, non-precipitating anti-VIII were separately bound covalently to CNBr-activated Sepharose. The rabbit antibody had a high affinity for VIIIR: AG, but a low affinity for VIII:C and VIIIR:WF. Passage of human plasma over the rabbit antibody column completely removed VIIIR:AG, but not the VIII:C or VIIIR:WF. The VIIIR:AG-free plasma was then sent over the human antibody column which removed only VIII:C. The effluent retained 60% of the original VIIIR:WF activity but had no measurable VIII:C or VIIIR:AG. Bio-Gel A-15 filtration of the VIIIR:WF resulted in elution of the activity in the Vo fractions. These data suggest that the VIII:C, VIIIR: AG and VIIIR:WF activities may exist in plasma as separate entities.