Three new proteins, FmhA, FmhB and FmhC, with significant identities to FemA and FemB were identified in the Staphylococcus aureus (ATCC 55748) genome database. They were mapped to the SmaI-C, SmaI-H and SmaI-A fragments of the S. aureus 8325 chromosome, respectively. Whereas insertional inactivation of fmhA and fmhC had no effects on growth, antibiotic susceptibility, lysostaphin resistance, or peptidoglycan composition of the strains, fmhB could not be inactivated, strongly suggesting that fmhB may be an essential gene. As deduced from the functions of FemA and FemB which are involved in the synthesis of the peptidoglycan pentaglycine interpeptide, FmhB may be a candidate for the postulated FemX thought to add the first glycine to the nascent interpeptide.