A two stage method for the determination of prothrombin is described in which the potent synthetic thrombin-inhibitor MD805 is used to suppress the inactivation process of thrombin by antithrombins in plasma. In the activation stage, prothrombin was activated almost instantaneously after the addition of tissue thromboplastin in 20-times diluted plasma and then the generated thrombin was inactivated progressively. Addition of MD805 suppressed the inactivation process of thrombin in a dose-dependent manner, while it hardly affected the complete activation of prothrombin. Thus, the generated thrombin was maintained stable in the presence of 50 microM MD805, and was measured by using 0.5 mM S-2238 as substrate. The method exhibited a linear relationship to the dilution of plasma. The decreased level of the other extrinsic coagulation factors such as FVII, FV, FX and tissue thromboplastin did not cause any significant changes until their activities were decreased to 3, 12, 12 and 20% of their controls. In addition, the method is very simple and easy for standardization.