Human factor VIII was purified from cryoprecipitate and subjected to enzymatic degradation with plasmin, trypsin, chymotrypsin, and thrombin. The reactions were monitored by sodium dodecyl sulfate (SDS)-Polyacrylamide gel electrophoresis and by measurement of four biologic properties, namely, procoagulant activity, ristocetin cofactor activity, precipitation by heterologous antibody, and neutralization of a human factor VIII inhibitor. The electrophoretic patterns of reduced digests showed a reproducible sequence of degradation of the 215,000 molecular weight subunit by plasmin, trypsin, and chymotrypsin, each with well-defined intermediate and enzyme-resistant chain remnants. There were striking similarities in the sizes of several intermediate chain remnants produced by these three enzymes, suggesting the presence of regions on the factor VIII molecule that are especially susceptible to enzymatic degradation, but each enzyme also produced distinctive proteolytic derivatives. The most obvious differences included (1) a single-chained 40,000 molecular weight remnant cleaved by plasmin and trypsin which was either absent or degraded in chymotryptic digests, (2) large intermediate chain remnants of 176,000 and 157,000 molecular weight which were produced only by trypsin, and (3) differences in size and content of nonreduced fragments in the final digests. Thrombin caused no detectable change in electrophoretic patterns. The immunoprecipitation reaction was sensitive to minor degrees of proteolytic cleavage, showing a marked increase in rocket size even when the SDS-treated subunit chains showed virtually no change in electrophoretic mobility. Procoagulant and inhibitor neutralizing activities were rapidly destroyed by trypsin and plasmin, but this destruction did not correlate with specific changes in electrophoretic patterns. Both thrombin and chymotrypsin caused an increase in procoagulant activity prior to destruction of activity; changes in this property were therefore unrelated to changes in the protein visualized by gel electrophoresis. Ristocetin cofactor activity was considerably more resistant to plasmin than to trypsin or chymotrypsin, and roughly paralleled the persistence of highly aggregated forms in the digests.