Purified bovine prothrombin was acetylated to a range of 27 to 30% of its amino groups. Except for just significant amounts it lost its power to generate thrombin-C. Upon activation the yield of thrombin-E was 100% on the basis of the thrombin-C yield that was obtained on an aliquot sample of the prothrombin before acetylation. Activation was achieved in 25% sodium citrate solution, with calcium ions plus brain extract thromboplastin, and with calcium ions plus protein-free brain thromboplastin. Evidently no Ac-globulin was needed; however, a concentrate of Ac-globulin accelerated the activation. The pH optimum for activation with brain extract thromboplastin is more to the alkaline side than with prothrombin. Activation with purified platelet factor 3 was quite slow, but was accelerated by adding a small amount of thrombin-E, in the form of acetylated thrombin, at zero time. Thrombin-C did not function in that capacity. A change in prothrombin had to be accompanied by a proportionate change in the thrombin to have the latter serve as a catalyst in the activation. Ac-globulin accelerates the hydrolysis of p-toluenesulphonyl-L-arginine methyl ester by thrombin. Ac-globulin owes its activity to thrombin. The view is expressed that Ac-globulin is measured as accelerated thrombin activity in the activation of prothrombin. In other words Ac-globulin accelerates the activation of acetylated prothrombin by thrombin-E. The amino groups on the N-terminal glutamic acid residues are free in the thrombin-E obtained from acetylated prothrombin. It is possible to acetylate an amino group in prothrombin that is necessary for thrombin-C activity.