A previously unrecognized penicillin binding protein (PBP) gene, pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and β-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed in Escherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region of pbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.