A membrane fraction from Pseudomonas aeruginosa KM 338 was shown to catalyze in vitro peptidoglycan synthesis from uridine 5'-diphosphate-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine and uridine 5'-diphosphate-N-acetylglucosamine. Synthesized peptidoglycan was partially cross-linked by transpeptidation, which was accompanied by the release of D-alanine. This reaction was strongly inhibited by 25 and 50 ,ug of penicillin G and carbenicillin per ml respectively, whereas the intact cells were relatively resistant to penicillins (minimal inhibitory concentration of penicillin G and carbenicillin, 30 and 0.125 mg/ml, respectively). Soluble D-alanine carboxypeptidase present in P. aeruginosa KM 338 was studied as well, which was found almost completely inhibited by penicillin G and carbenicillin (10