Factor VIII is a large protein molecule of molecular weight 2,000,000 or larger that elutes in the void volume on agarose gel chromatography. It has been shown previously that high concentrations of alkali halides and, more specifically, 0.25 M Ca(2+) dissociate the molecule into a large carrier protein and a small fragment that retains the factor VIII activity. Factor VIII was prepared from normal canine plasma collected in sodium oxalate and heparin and adsorbed with BaSO(4). Results with Ca(2+) dissociation were the same as those obtained with fraction prepared from canine plasma collected in sodium citrate. The addition of 0.1 M epsilon-aminocaproic acid in the dissociation step had no effect. Fractionation of canine hemophilic plasma produced preparations without activity, and no activity was found when these inert preparations were dissociated with Ca(2+). These results indicate that the Ca(2+) dissociation is a true dissociation and not caused by enzymatic degradation by plasmin, thrombin, or activated factors VII, IX, or X. The apparent molecular weight of the small active fragment of factor VIII determined by gel chromatography was about 100,000. Finally, when the large carrier protein and the small active fragment of factor VIII were separated by gel chromatography, mixed, and dialyzed free of Ca(2+), they recombined to form a large active molecule that appeared in the void volume on agarose gel chromatography.