The purfied antibacterial cationic proteins 1 to 7 (component 1 being the most cationic) of human neutrophil granulocytes have been assayed for enzyme activities. Components 1 to 4 and component 7 exhibited activities on tyrosine esters indicating a chymotrypsin-like activity with a neutral pH optimum. The inhibition of the esterase activity by diisopropyl fluorophosphate and phenylmethyl sulfonylfuoride also indicated the similarity to chymotrypsin, with a serine residue in the active center. In addition, the cationic proteins attacked complex substrates like casein and fibrinogen, suggesting a true proteolytic activity. Since the esterase activity of these proteins is heat-labile but the antibacterial activity against Staphylococcus aureus is heat-stable, it is concluded that the chymotrypsin-like activity is not essential for the microbicidal activity. There is evidence to suggest that the cationic proteins of human granulocytes are present in the cytoplasmic granules and exhibit antibacterial activity after release to the phagocytic vacuole and that, after extrusion from the cell, the proteins might be involved in the mediation of the inflammatory process due to enzyme and cationic activities.