Actinobacillus pleuropneumoniae, which was formerly classified in the genus Haemophilus, is a pathogen causing swine pleuropneumonia. We found that aspoxicillin showed strong activity and that meropenem had better lytic activity against this pathogen. In the present study, we for the first time identified penicillin-binding proteins (PBPs) of A. pleuropneumoniae in order to elucidate the relationship between the antibacterial and lytic activities of β-lactam antibiotics and affinities of the PBPs. The competitive assay using 3H-labeled benzylpenicillin revealed seven PBPs in A. pleuropneumoniae; they were determined to be PBPs 1a, 1b, 2, 3, 4, 5, and 6, and the molecular masses of these PBPs were estimated to be 92, 80, 76, 72, 50, 44, and 30 kDa, respectively, by comparison with those of Haemophilus influenzae. Our detailed analysis of the affinities of the PBPs of A. pleuropneumoniae and of the bacterial lysis kinetics for several β-lactam antibiotics revealed that the strong antibacterial activity of aspoxicillin against this strain could be related to the higher affinity of PBP 3 and that preferential inactivation of PBP 1b could cause rapid lysis.