Protamine, a highly purified basic polypeptide of 4000 molecular weight containing 80-85% arginine. is a useful substrate for the assay ofplasmin. activated plasminogen, and enzymes of similar specificity, e.g., urokinase, coagulation factor Xa, trypsin, and thrombin, and is also an excellent secondary substrate for activator assays of urokinase and streptokinase. The assays were performed manually, and also automated procedures for continuous multiple sample analyses were used. The relative sensitivities for various plasmin-like enzymes were: trypsin > plasmin > urokinase > factor Xa > thrombin. Using protamine with manual assay procedures, the amino-terminal groups of the enzyme-degraded protamine digestion products were detected and quantitated by the calorimetric ninhydrin or the fluorometric fluorescamine procedures, and using protamine with an automated system the ninhydrin method was used. Assigning the CTA casein assay for plasmin a nominal sensitivity of 0.1 (for 0.1 CTA unit of plasmin). the sensitivities of the various assay methods were casein. automated protamine, and manual protamine with ninhydrin, 0.1; manual protamine-trichloroacetic acid with fluorescamine, 0.005; and manual protamine direct fluorescamine, 0.0005. A unit of plasmin, based on the uptake of 1 pequiv base/min during hydrolysis of 0.4% protamine sulfate under standard conditions, is equal to approximately 1.7 x 103 RF1 units or 2.9 CTA units; or, 1 CTA unit ofplasmin resulted in an average uptake of 0.346 pmol of base or equivalent bonds split per minute.