A kininogenase (bradykinin-releasing enzyme) from the venom of A. caliginosus, is a single polypeptide-chain glycoprotein with a mol.wt of about 33,500, which contains 10.1% carbohydrate. The isoelectric point of the enzyme is 3.5 and the enzyme has 274 amino acid residues based on the mol.wt of 33,500. The enzyme hydrolyzed arginine esters more readily than lysine esters, but did not hydrolyze tyrosine ester. The activity of the enzyme on hydrolysis of arginine ester or on liberation of kinin from purified bovine high mol.wt kininogen was inhibited by diisopropylfluorophosphate, indicating that the serine hydroxyl group is involved in enzymatic activity. Moreover, the enzyme split N-alpha-carbobenzoxy-Gly-Pro-Arg-p-nitroanilide (PNA), H.D.Val-Leu-Arg-PNA, H.D.Pro-Phe-Arg-PNA, H.D.Phe-pipecolyl-Arg-PNA and Pro-Phe-Arg-4-methylcoumaryl-7-amide more readily than the other chromogenic or fluorogenic substrates. This result indicates that the substrate specificity of the enzyme is broader than that of mammalian serine proteinases.