N alpha-Benzyloxycarbonyl-p-guanidino-L-phenylalanine beta-naphthylamide (Z-GPA-beta NA) was synthesized and the susceptibility of this compound to trypsin and related enzymes was compared with that of N alpha-benzyloxycarbonyl-L-arginine beta-naphthylamide (Z-Arg-beta NA). Both Z-GPA-beta NA and Z-Arg-beta NA were rapidly and almost completely hydrolyzed by trypsin and pronase. Z-Arg-beta NA was hydrolyzed slowly by thrombin, while Z-GPA-beta NA was not susceptible to this enzyme at all. The rate of hydrolysis of Z-GPA-beta NA by papain was slower than that of Z-Arg-beta NA. Neither beta-naphthylamide substrate was hydrolyzed by alpha-chymotrypsin. The specificity constant (kcat/Km) for the hydrolysis of Z-GPA-beta NA by trypsin was somewhat larger than that for the hydrolysis of Z-Arg-beta NA. Contributions of the benzene ring in the side chain of Z-GPA-beta NA to good binding of this substrate to the specificity site of this enzyme and to the poor fit of the scissile bond in the substrate molecule to the active serine residue are presumed from comparison of the individual kinetic parameters (Km and kcat) for the two beta-naphthylamide substrates. Z-GPA-beta NA was ascertained to be a useful substrate in the study of the binding and catalytic specificities of various trypsin-like enzymes.