In order to study the enzymatic properties of α-amino acylase of KT 801 (Pseudomonas sp.), which has δ-ornithine acylase, purification of the α-amino acylase have been attempted. α-Amino acylase of KT 801 was extracted by means of spheroplast formation and purified by fractionation with ammonium sulfate, acrinol treatment, and CM Sephadex chromatography. The specific activity of the purified enzyme toward N-benzoyl-L-alanine was 2500 μM/hr/mg and it was represented about 70 fold purification over the original cell suspension. This enzyme has a pH optimum around 8.0 toward benzoyl-L-alanine. It has an optical specificity and hydrolyzes benzoyl-L-amino acids. Also, it hydrolyzes α-N-benzoyl-L-amino acids but not phenylacetyl amino acids. The ratio of the activities toward α-N-benzoyl amino acids remained constant all through the purification steps except for 2-N-benzoyl-L-ornithine. These results suggest that one enzyme hydrolyze these α-N-benzoyl amino acids. Then, the α-amino acylase of KT 801 was provisionally named "benzoylamino acid amidohydrolase."