An improved XIEP technique has been described that is capable of resolving under nondenaturing conditions at least five populations of FVIIIR:Ag directly from microliter amounts of normal human plasma. Resolution has been achieved by increasing the concentration of agarose in the electrophoretic medium to 2% or 3% and extending the period of electrophoresis to take advantage of molecular sieving effects. Normal human plasma demonstrates an electrophoretic pattern indicating that the majority of FVIIIR:Ag is assembled as higher-molecular-weight multimers. The distribution of FVIIIR:Ag appears to be independent of antigen concentration and sample storage temperature. Plasmas from two individuals with variant VWD demonstrate a lack of the higher-molecular-weight multimers coupled with an absolute increase in the lower-molecular-weight antigenic populations, suggesting a defect in antigen assembly. Commercial FVIII preparations, reported to be ineffective in correcting the bleeding abnormality in VWD, have also been found to lack the higher-molecular-weight multimers. On the other hand, cryoprecipitate, preferred in the treatment for VWD, had essentially the same antigen distribution as normal plasma. The similarities in the distribution of FVIIIR:Ag in variant plasmas and commercial FVIII concentrates provide further evidence that it is primarily the higher-molecular-weight multimers that function in maintaining hemostasis.