It has been reported by Kameda et al (1) that three different kinds of acylases capable of performing the resolution of acyl-DL-amino acids were produced from a Pseudomonas species, KT-83, which was previously isolated from soil (2), and that two of those acylases hydrolyze asymmetrically the acyl groups at α- and ε-position of Nα, Nε-dibenzoyl-DL-lysine, and the third one hydrolyzes the acyl group of Nα-benzoyl-D-phenylalanine. Whereas there have been published many papers dealing with the isolation of the α-lysine acylases from Aspergillus species (3) hog kidney (4) and other sources (5), only one report has been so far available on the ε-lysine acylase by Paik et al. (6) who obtained it from rat kidney. In contrast to the fact that these acylases are able to hydrolyze acetyl-DL-amino acids, and are practically ineffective upon benzoyl-DL-amino acids, the acylases prepared from KT-83 asymmetrically hydrolyze benzoyl-DL-amino acids in preference to acetyl-DL-amino acids. The author has undertaken the fractionation of the crude ε-lysine acylase of KT-83 (supplied by K a m e d a) and has obtained the enzyme preparation as four thousand fold active as the original enzyme solution. The highly purified enzyme possesses only ε-lysine acylase activity. A systematic study of the enzymatic activity at various stages of purification has clarified the mechanism of the conversion by KT-83 of Nα, Nε-diacyl-DL-lysine to L-lysine and unchanged Nα, Nε-diacyl-D-lysine.