The thrombogenicity of three highly purified proteases (thrombin, activated Factor X, and activated Factor IX) was determined quantitatively in an animal model. The minimal amounts required to produce a standard score 4 thrombus were 1.1 nmol for thrombin, 0.12 nmol for activated Factor X, and 0.018 nmol for activated Factor IX. After the administration of heparin at 5, 10, and 20 units/kg in rabbits, the thrombogenicity of each of these proteases decreased progressively. The heparin-induced inhibition of thrombosis decreased in the order, activated Factor IX > activated Factor X > thrombin at each heparin concentration. These differences were statistically significant. These in vivo data provide support for the following hypotheses originally developed from in vitro experiments: (i) activation of the blood coagulation system, which proceeds through a cascade mechanism, incorporates biochemical amplification; (ii) the inhibition of activated Factor IX by antithrombin III in the presence of heparin is an important reaction in the prevention of thrombosis; (iii) less heparin is required to inhibit thrombosis prior to thrombin generation than afterward; (iv) an increase in the reactivity of antithrombin III reflects a decreased tendency to thrombosis while a decrease in this reactivity reflects an increased tendency to thrombosis.