Inactivation of femC in methicillin-resistant Staphylococcus aureus (MRSA) results in lowered methicillin resistance and a reduction in the amidation of the iso-D-glutamate of the peptidoglycan stem peptide. The femC phenotype is due to insertional inactivation of the glutamine synthetase repressor gene glnR by Tn551, which has a polar effect on glutamine synthetase (glnA) transcription. The complete glutamine synthetase operon (glnRA) of S. aureus was cloned and sequenced, and its transcriptional start was determined. The deduced amino acid sequence of the staphylococcal glutamine synthetase showed 76% identity and 87% similarity to the Bacillus subtilis glutamine synthetase. The staphylococcal glnRA operon was shown to complement an Escherichia coli glutamine synthetase-negative mutant and to restore methicillin resistance in femC mutants. femC mutants revert to resistance in the presence of high concentrations of methicillin. These revertants, which still carried the femC lesion, were shown to retain the lowered amidation of the iso-D-glutamate peptidoglycan stem peptide. A new chromosomal locus hmrC was postulated to have mutated to allow expression of high methicillin resistance in these femC revertants. Although the highly resistant hmrC revertant resembled phenotypically the highly methicillin-resistant subclones occurring in heterogeneously resistant MRSA, we could show by transduction that the locus hmrC was distinct from chr*, a chromosomal site postulated to confer high methicillin resistance in heterogeneous MRSA. This suggests that S. aureus can adopt multiple ways to achieve high methicillin resistance.