The penicillin-binding protein (PBP) profiles of 33 Clostridium perfringens and six Clostridium species isolated from clinically significant infections were analyzed. Three new PBPs-PBPs 2B, 4B, and 5B (84, 70, and 49 kDa respectively)-and a high-molecular-weight PBP 6 (45 kDa) were demonstrated in the C. perfringens isolates. In addition to PBPs 1 and 2, PBPs 2B and 4B were seen to show low binding affinities for penicillin, although further studies are required to determine their possible roles in the development of penicillin resistance. The PBP profiles of the C. perfringens isolates were complex. Variations in apparent molecular weights (M(r)s) of all PBPs, with the exception of PBP 5 and the presence or absence of PBPs 2, 3, and 4B, gave rise to nine different PBP patterns. The high-M(r) PBPs 5 and 6, which exhibited high-penicillin-binding affinities, were with only one exception consistent within the C. perfringens isolates. These PBPs 5 and 6 of the C. perfringens isolates and independent PBPs found in the other Clostridium species studied indicate that PBP analysis may assist in the differentiation of Clostridium species.