The aminopeptidase P gene in Escherichia coli HB1O1 was cloned into the plasmid pBR322. Introduction of the hybrid plasmid, pAPPO1, into the E. coli DH1 resulted in an 8-fold increase of aminopeptidase P activity as compared with that of the host. The enzyme was purified by series of chromatographies on DEAE-Sephadex, QAE-Sephadex, and hydroxy apatite. The purified enzyme was homogeneous as judged by disc-gel and SDS-gel electro phoreses. The enzyme was inhibited strongly by EDTA and slightly by p-chloromercuri benzoate, but was not affected by diisopropyl phosphorofluoridate, E-64, or iodoacetic acid. The optimum pH of the enzyme was 8.5. The enzyme was stable at pH 8 to 9. After incubation for 30 mm at pH 8.0, 50% remaining activity was observed at 50° The enzyme was activated 3-fold by the addition of 5 μM Mn²⁺ The molecular weight of the enzyme was estimated to be 50,000 and 200,000 by SDS-PAGE and gel filtration, respectively. The amino terminal amino acid was identified to be serine by Edman degradation, indicating that the enzyme is composed of a homo-tetramer. The enzyme hydrolyzed X-Pro bonds (X = amino acid) of peptides. These characteristics suggest that cloned aminopeptidase P is identical to APP-II reported by Yoshimoto et al. (Agric. Biol. Chem. 52(8), in press (1988).