The bovine coagulation factors II, VII, IX and X were separated from other plasma proteins, and each factor was obtained in a largely pure state by various chromatographic procedures in the presence of diisopropylfluorophosphate (DFP). Factor X was separated from factors II, VII, and IX by DEAE Sephadex chromatography, followed by G-100 filtration and hydroxylapatite chromatography. Separation of factors II and IX from factor VII was achieved by chromatography on DEAE cellulose, followed by G-100 gel filtration and hydroxylapatite chromatography. Factors II and IX were separated by preparative polyacrylamide electrophoresis. After DEAE cellulose chromatography, factor VII was purified by gradient elution from hydroxylapatite.