The degradation of bovine Factor VIII by plasmin, trypsin, and thrombin was monitored with polyacrylamide gel electrophoresis in a urea—acetic acid system. The sequences of plasmic and tryptic proteolysis were very similar, proceeding via several intermediate, enzyme-sensitive products to smaller derivatives which were relatively resistant to further degradation. The intermediate degradation products were similar for both enzymes, although prolonged digestion with trypsin produced final products which were smaller than those observed with plasmin. Stages of digestion which reflect the degree of completeness of proteolysis were defined on the basis of the presence or absence of these distinct components in the electrophoretic profiles. Thrombin treatment of Factor VIII caused a loss of coagulant activity but did not yield products small enough to be characterized in this electrophoretic system. During the course of plasmic and tryptic digestion, there was a rapid loss of all coagulant activity without the generation of anticoagulant activity. Agarose chromatography of a 24-hr plasmic digest revealed three major protein peaks, which corresponded to individual electrophoretic bands on polyacrylamide gel electrophoresis in a nondenaturing system at pH 8.8. Upon polyacrylamide gel electrophoresis in the urea—acetic acid system, the components of the first two of these peaks dissociated into two and three separate fragments, respectively. Immunoelectrophoresis demonstrated two major distinct antigenic determinants in these first two elution peaks.