A method for isolation of factor VIII from cryoprecipitate fraction of human plasma has been elaborated. The isolation procedure involves precipitation with dextran, removal of fibrinogen by means of defibrase, precipitation with ammonium sulfate, polyethylene glycol fractionation, and Sepharose 6B gel filtration step. Factor VIII has been purified 7000- to 13,000 -- fold.. The preparation is homogenous by ultracentrifugal examination and it has an S20,w value of 19.4. It also shows a single precipitin line when subjected to immunoelectrophoresis employing rabbit antibodies against factor VIII. The preparation did not enter a 7.5% polyacrylamide gel containing sodium dodecyl sulfate even in the presence of 8 M urea. After reduction of the protein with 2-mercaptoethanol, subunits were formed which migrated as one band in polyacrylamide gel electrophoresis.