The mechanism of action of a new antipseudomonal penicillin, PC-904, was studied with respect to its binding affinities to penicillin-binding proteins (PBPs) and its inhibitory activities on cross-linking enzymes of peptidoglycan synthesis in vitro. PC-904 showed especially high affinity (compared with that of penicillin G) to Escherichia coli PBP-3. It also had high affinities to PBP-2 and -1Bs and low affinities to PBP-1A, -4, -5, and -6. Similar results were obtained with Pseudomonas aeruginosa, in which this antibiotic showed very high affinity (compared with that of penicillin G) to PBP-3, -1A (presumably corresponding to E. coli PBP-1Bs), and -2; there was especially high affinity to PBP-3 and much less affinity to PBP-1B (presumably corresponding to E. coli PBP-1A). These results are compatible with morphological observations that at concentrations near its minimal inhibitory concentration or less, this antibiotic induced the formation of filamentous cells of E. coli and P. aeruginosa. At higher concentrations or after prolonged incubation, it induced lysis of the cells. The remarkably high affinity of PC-904 to pseudomonal PBP-3, -1A, and -2 may partly explain the potent antipseudomonal activity of this antibiotic. In E. coli, the concentration of PC-904 required to inhibit the cross-linking reaction in enzymatic peptidoglycan synthesis, presumably carried out by PBP-1Bs, was as low as the inhibitory concentrations of penicillin G, ampicillin, and carbenicillin.