The potential for the development of resistance in oxacillin-resistant Staphylococcus aureus (ORSA) to lysostaphin, a glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus, was examined in vitro and in an in vivo model of infection. Following in vitro exposure of ORSA to subinhibitory concentrations of lysostaphin, lysostaphin-resistant mutants were idenitifed among all isolates examined. Resistance to lysostaphin was associated with a loss of resistance to β-lactams and a change in the muropeptide interpeptide cross bridge from pentaglycine to a single glycine. Mutations in femA, the gene required for incorporation of the second and third glycines into the cross bridge, were found following PCR amplification and nucleotide sequence analysis. Complementation of lysostaphin-resistant mutants with pBBB31, which encodes femA, restored the phenotype of oxacillin resistance and lysostaphin susceptibility. Addition of β-lactam antibiotics to lysostaphin in vitro prevented the development of lysostaphin-resistant mutants. In the rabbit model of experimental endocarditis, administration of a low dose of lysostaphin for 3 days led predictably to the appearance of lysostaphin-resistant ORSA mutants in vegetations. Coadministration of nafcillin with lysostaphin prevented the emergence of lysostaphin-resistant mutants and led to a mean reduction in aortic valve vegetation counts of 7.5 log10 CFU/g compared to those for untreated controls and eliminated the isolation of lysostaphin-resistant mutants from aortic valve vegetations. Treatment with nafcillin and lysostaphin given alone led to mean reductions of 1.35 and 1.65 log10 CFU/g respectively. In ORSA, resistance to lysostaphin was associated with mutations in femA, but resistance could be suppressed by the coadministration of β-lactam antibiotics.