To evaluate the extent of denaturation of factor VIII-coagulant activity (VIII: C) during production of factor VIII concentrates, the factor VIII-coagulant antigen (VIII: CAg)/VIII: C ratio was measured in plasma, cryoprecipitate and cryosupernatant from fresh and stored blood. This ratio was close to unity for both cryoprecipitate and other concentrates, suggesting that VIII: CAg is lost concurrently with VIII: C during cryoprecipitation and further fractionation. Storage of blood (18 h, 22 degrees C) before processing resulted in a 30% loss of VIII: C from the separated plasma; however, VIII: CAg was not affected. In cryoprecipitate prepared from this plasma, VIII: C and VIII: CAg both were 30% lower than when prepared from fresh plasma. In the corresponding cryosupernatant, however, more VIII: CAg but less VIII: C was present compared with fresh material. Gel chromatography revealed that the rise of VIII: CAg in cryosupernatant prepared from stored blood, was due to an increased amount of VIII: CAg of low molecular weight, not being associated with factor VIII-related antigen. Such an increase in dissociated VIII: CAg was not detected in the plasma prior to cryoprecipitation. It is concluded that during storage of blood, molecular changes are induced in the factor VIII-VWF complex, possibly by limited proteolysis, which make the complex more liable to dissociation during subsequent cryoprecipitation.