Prothrombin preparations were examined by electrophoresis and found to contain approximately 90 per cent of the protein in one component. Such products possess a specific activity of 1,400 units per milligram of dry weight when analyzed with reagents and technics that give a value of 32.9 ± 30 units of prothrombin per cc. of oxalated bovine plasma. The electrophoretic mobility is approximately equivalent to that of α1-globulin. That of thrombin is less. The isoelectric point in 0.1 ionic strength salt solution is near pH 4.2, and for thrombin it is approximately pH 4.8. Analysis of dry preparations indicated 0.96 per cent sulfur, 13.57 per cent nitrogen, 4.58 per cent tyrosine, and 3.33 per cent tryptophane, and no phosphorus was found. The purified material could be activated to thrombin by dissolving the prothrombin in concentrated solutions of sodium citrate, ammonium sulfate, magnesium sulfate, sodium sulfate, potassium citrate, potassium oxalate, and dipotassium hydrogen phosphate. Concentrated solutions of sodium chloride, potassium chloride, ammonium chloride or magnesium chloride do not produce activation. Difficulties were encountered when attempts were made to activate less highly purified prothrombin preparations with sodium citrate. The activation with sodium citrate is autocatalytic, and the yield of thrombin can be increased to a maximum by adding a small amount of 3-methyl-4,6,4'-triaminodiphenyl sulfone to the activation mixture. The sodium citrate concentration must be high, preferably near 25 per cent. A new method for preparing thrombin is based on the use of these principles. During activation the electrophoretic properties of prothrombin change long before appreciable amounts of thrombin activity are found. These changes in electrophoretic properties are associated with a prothrombin derivative which is refractory to the action of the activators calcium plus thromboplastin plus Acglobulin. When activation with sodium citrate is complete almost none of the material which had electrophoretic mobility of prothrombin is found in the electrophoresis boundary patterns. The major portion of the pattern is comprised of two components having mobilities less than prothrombin while a third component with high mobility is especially evident in the descending boundary pattern. Solubility studies indicate that two proteins may possess thrombin activity, but the evidence is not conclusive. An attempt was made to separate the proteins in the thrombin preparation by fractionation with ammonium sulfate, and it was found that their properties are so similar that this procedure is not effective. Thrombin obtained by activation of prothrombin with sodium citrate may be dried from the frozen state and is stable. Prothrombin is not stable after drying in this manner; however, it can be dried with acetone. It is reasoned that since sodium citrate and other salts can produce thrombin from prothrombin, the latter must contain all the structural material needed for the thrombin. For that reason calcium, thromboplastin, and Ac-globulin are regarded as catalysts of prothrombin activation and not as activators which must contribute material substance in prothrombin activation.