The penicillin-binding proteins (PBPs) of Clostridium perfringens were studied. Six PBPs ranging in molecular weight from approximately 42,000 to 100,000 were detected in the cytoplasmic membrane. The relative affinities of the PBPs for 16 beta-lactam antibiotics were determined. Most of the drug saturated PBP 3 and 4 at concentrations equal to their minimal inhibitory concentrations, suggesting that these PBPs are the killing targets for beta-lactams. Mecillinam showed unique properties; it had a higher affinity for PBP 5 than for other PBPs, and it was the only agent tested which caused inhibition of growth without saturating PBP 4. Interestingly, all beta-lactam antibiotics tested induced filament formation despite having different binding patterns to the PBPs of C. perfringens.