A beta-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined beta-peptidyl aminopeptidase of Pseudomonas aeruginosa PAO1 (BapF) to assess the effects of mutation of catalytic Ser with Cys or Thr on its catalytic ability. Recombinant BapF and its S237C mutant exhibited p-nitroaniline release activity toward beta-homo-Gly-p-nitroanilide (beta hGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed beta hGly-beta hGlypNA synthetic activity, but the product vanished in a few minutes and converted to free beta hGly. In contrast, the product beta hGly-beta hGly-pNA was synthesized by S237C BapF efficiently without degradation, indicating that because of the mutation, the enzyme came to recognize only the amine group as an acyl acceptor instead of water. Furthermore, a difference in acyl acceptor preference between that of wild type and S237C BapF was observed. When using cysteamine as an acyl acceptor, beta hGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize beta hGly-cystamine when using cystamine as an acyl acceptor, although it was synthesized by wild-type BapF. Such a dynamic change in the acyl acceptor by the mutation of catalytic Ser with Cys is regarded as a unique feature of family P1 peptidases.