Fibrinolytic inhibitor was prepared from human aortas and some of its biochemical properties were investigated. The fibrinolytic inhibitor suppressed urokinase activity, but did not inhibit plasmin activity when assayed by fibrin plate method and synthetic fluorogenic substrate method. The urokinase inhibitor was a glycoprotein and migrated similar to alpha-globulin upon fibrin-agar electrophoresis. The molecular weight determined by gel filtration was approximately 98,000. The urokinase inhibitor was immunologically different from other known plasma protease inhibitors, such as alpha 2-plasmin inhibitor, alpha 2-macroglobulin, and alpha 2-antitrypsin. The interaction of urokinase with the inhibitor was dose-dependent. Progressive inactivation of urokinase occurred by increasing time of incubation with the inhibitor at 37 degrees C, and over 90% inhibition of urokinase required 30 min of incubation. The inhibitor of plasminogen activator in human aorta may be noteworthy in relation to thrombogenesis and atherogenesis.