Purified prethrombin was converted to thrombin in an activating system consisting of purified autoprothrombin C, purified Ac-globulin, lipid and calcium chloride. With the concentration of calcium and pH fixed, the effect of varying the other 3 pro-coagulants was studied. Conditions influencing the conversion of prethrombin to thrombin were determined. Autoprothrombin C is the enzyme, but with it alone activation was very slow. Best results were obtained with the simultaneous presence of autoprothrombin C, Ac-globulin and lipid. Reducing any one of the three toward zero concentration decreased the rate and yield of thrombin generation. In association with rapid thrombin generation Ac-globulin and autoprothrombin C were represented in approximately a 1:1 molar ratio. In that combination of Ac-globulin and autoprothrombin C the Michaelis constant for autoprothrombin C with prethrombin as a substrate was 3.14 × 10-6M. * This investigation was supported by a research grant HE-03424-09 from the National Heart Institute, National Institutes of Health, IT. S. Public Health Service.