We detected a novel protein with [14C]benzylpenicillin (PCG)-binding capacity and a molecular mass of about 60 kD in methicillin-resistant Staphylococcus aureus using a high concentration of [14C]PCG and extending the reaction time. However, the fluorogram showed that the band density of penicillin-binding protein 2′ (PBP2′) decreased gradually with incubation time. The appearance of the 60-kD protein and the reduction of the band density of PBP2′ were stoichiometrically linked, and the binding profiles of β-lactams for PBP2′ and the 60-kD protein corresponded. These results suggested that the 60-kD protein is a degradation product