A Tn551 insert in a gene termed fmtB was shown to reduce oxacillin as well as Triton X-100 resistance in highly methicillin-resistant Staphylococcus aureus (MRSA) COL. Backcrosses of fmtB::Tn551 into S. aureus COL and into two genetically distinct MRSA strains, KSA8 and NCTC10443, confirmed the linkage of fmtB::Tn551 with loss of oxacillin resistance. The fmtB gene codes for a protein of a deduced molecular mass of 263 kDa that contains 17 tandem repeats of 75 amino acids and a C-terminal LPXTG cell wall-sorting motif. Immunoblots with anti-FmtB antibodies confirmed its localization in the cell wall fraction. The fmtB gene was mapped downstream of the phosphoglucosamine mutase operon glmM which catalyses formation of glucosamine-1-phosphate. Oxacillin resistance was not restored in fmtB mutants by trans-complementation with fmtB. However, although GlmM production was not affected by fmtB inactivation, oxacillin resistance was increased in fmtB mutants by introducing a plasmid-borne glmM gene, presumably by GlmM overexpression. Interestingly, a similar phenotypic complementation was obtained in fmtB mutants by including substrate level concentrations of N-acetylglucosamine or glucosamine in the growth medium. Inactivation of the fmtB gene seems therefore to have an indirect effect on methicillin resistance which can be relieved by increasing the production of the cell wall precursor glucosamine-1-phosphate.