ABSTRACTTwo electrophoretically homogeneous proteinases designated proteinase A and B were isolated from anchovy viscera. Purity was increased 17.7 and 24.6‐fold with approximately 1.9 and 1.8% yield for proteinases A and B, respectively. The maximum caseinolytic activity was found to be at pH 9.4 for proteinase A and at pH 9.6 for proteinase B at the optimum temperature of 48°C. The molecular weights of proteinase A and B were determined to be 27, 300 and 25,100 D, respectively, using Sephadex G‐100 gel filtration. The amino acid profiles of the enzymes were similar and relative proportion of amino acid residues was comparable to that in bovine pancreatic α‐chymotrypsin. Proteinase A and B were identified as α‐chymottypsin‐like serine proteases by inhibitor and substrate specificity studies. Apparent Km(Km′) values of proteinase A and B for benzoyl‐L‐tyrosine ethyl ester were 4.6 × 10−4M and 1.2