Highly purified human plasminogen was activated by urokinase to plasmin (EC 3.4.4.14), which was subsequently purified by Sephadex G-150 gel filtration. Purified human Plasmin was found to catalyze fibrinolysis, caseinolysis as well as hydrolysis of N-tosyl-L-arginine methyl ester. These activities were abolished by heat treatment. Purified human plasmin was inactivated much more slowly by TLCK (a chloromethylketone derivative of N-tosyl-L-lysine) than trypsin. Stability of purified plasmin was examined under various conditions.