The molecular weights of trypsin and chymotrypsin purified from anchovy viscera were estimated to be 25.6 and 26.1 Kda, respectively, by SDS-PAGE. Both enzymes had their maximal activity at pH 9.0 and 45°C for casein and at pH 8.0 and 45°C for synthetic substrates. Trypsin hydrolyzed at the position of Arg 2 2 and Lys 2 9 , and chymotrypsin did at the position of Phe 1 , Tyr 1 6 , Phe 2 4 , Phe 2 5 , and Tyr 2 6 of insulin β-chain. The K m and k c a t of trypsin were 50 μM and 1.84 μM - 1 min - 1 toward N-benzoyl-l-arginine-p-nitroanilide (BAPNA) and those of chymotrypsin were 89 μM and 10.0 μM - 1 min - 1 toward N-succinyl-(Ala) 2 -Pro-Phe-p-nitroanilide. The activation energy of trypsin and chymotrypsin were estimated to be 14 Kcal/mol toward N-benzoyl-l-arginine-p-nitroanilide and 6.5 Kcal/mol toward benzoyl-l-tyrosine ethyl ester.