Results relative to inhibitory effects and substrate specificity indicated that a protease from the muscle of anchovy, Engraulis japonica, was a cathepsin L-like enzyme. The enzyme was activated by thiol reagents and inhibited by thiol-blocking reagents. The molecular weight was estimated to be 25.8 kDa by SDS-PAGE. The enzyme exhibited its maximal activity at pH 6.0 and 50 degrees C for casein and N-benzoyl-D, L-arginine-beta-naphthylamide. The enzyme hydrolyzed at the position of Phe1, Asn3, Val13, Glu14, Val19 and Gly24 of the insulin beta-chain. The K'm and kcat of the enzyme were 73.4 microM and 0.5 microM/min, respectively, toward Z-Phe-Arg-MNap.