Hydrolysis of various substrates by proteolytic enzymes from human blood leukocytes was studied. Among the protein substrates used protamine was hydrolyzed at the highest rate. Among the synthetic substrates--N-benzoyl-D,L-arginine ethyl ester was the one hydrolyzed most rapidly. Effect of native inhibitors of proteinases and of bivalent metal ions on the proteolytic activity in leukocytes was also studied. The highest proteolytic activity, estimated by hydrolysis of protamine, was found in lysosomal and microsomal fractions.