Partially purified penicillin acylases (EC 3. 5. 1. 11) were prepared from Pseudomonas melanogenum KY 3987 and Kluyvera citrophila KY 3641 capable of synthesizing D (-)-α-aminobenzylpenicillin (APc) from 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester. As the cell-free extract of P. inelanogenum contained high levels of penicillinase (EC 3. 5. 2. 6), the acylase was separated completely from the penicillinase by use of Sephadex column chromatography or electrofocusing. The most salient property of the P. melanogenum penicillin acylase was its substrate specificity to penicillin substrates: it could form 6-APA only from APc but not from penicillin G, penicillin V and p-aminobenzylpenicillin, whereas the K. citrophila acylase acted on all of these penicillins. The P. melanogenum enzyme is hence considered a novel type of penicillin acylase.