Further selection for a better strain capable of producing D(−)-α-aminobenzylpenicillin (APc) from 6-aminopenicillanic acid (6–APA) was carried out. Pseudomonas melanogenum KY 3987 was consequently selected as a new strain possessing an APc-specific penicillin acylase.The acylase could synthesize APc in good yields from 6–APA and phenylglycine ester and form 6–APA only from APc, not from other common penicillins. Since the Pseudomonas acylase was found incapable of forming penicillin G (Pc–G) from 6–APA and phenylacetic acid, in contrast with E. coli and Kluyvera citrophila enzymes, the enzymatic hydrolysate of Pc–G, for example by K. citrophila cells, which contained 6–APA and phenylacetate, became employed as a source of 6–APA instead of purified 6–APA to synthesize APc by the cells of P. melanogenum.