The enzymatic resolution of acetyl-dl-methonine previously reported was studied further, in detail. As a result, it was found that metal ion plays an important role on the asymmetric hydrolysis of acyl-dl-methonines by the enzyme preparations of Aspergillus oryzae. The advantageous resolution procedure of dl-lysine previously reported, consisting of the enzymatic hydrolysis of ε-benzoyl-α-acetyl-dl-lysine with mold enzyme preparation was innovated. Susceptibility of ε-benzoyl-α-acetyl-dl-lysine towards various enzyme preparations such as kidney, pancreas extracts, renal acylase and carboxy-peptidase, was investigated. As a result, it was found that although the substrate was readily hydrolyzed by the mold enzyme preparations, the other enzyme preparations showed, almost no, if any, hydrolytic activity. The distribution of activity hydrolyzing acyl amino acids was investigated, so as to obtain an advantageous enzyme source for the resolution of amino acids. For this purpose, a number of molds such as Aspergillus, Penicillium, Rhizopus, Mucor, Abshidia, and Cunninghamella were cultivated on wheat bran and the extracts were tested in relation to their hydrolytic activity of acyl amino acids. Although almost all molds yielded acylase more or less, high activity was shown in cases of Aspergillus and Penicillium. As one of the most suitable strains, Aspergillus oryzae No. 9 was chosen and the optimum cultural Conditions for acylase formation was also inveitigated. With the mold acylase preparation, optimal pH and relative activity to the various acyl amino acids were investigated. As a result, a difference of optimal pH was observed between acetyl and chloroacetyl derivatives. A comparison of the susceptibility disclosed that mold preparation has either a wider or loose specificity than renal acylase. Namely, besides aliphatic amino acids, the acyl derivatives of basic amino acids and aromatic amino acids were readily hydrolyzed by the mold preparation. These results indicate that the mold acylase preparation can be used for the resolution of a great variety of amino acids. As an example of this, the resolution of acetyl-dl-tryptophan was presented.