Mechanisms responsible for the development of acquired resistance to lysostaphin, a glycylglycine endopeptidase, among isolates of Staphylococcus epidermidis were examined. Following overnight exposure to sub-inhibitory concentrations of lysostaphin, lysostaphin-resistant, oxacillin-susceptible mutants were isolated and characterized. Lysostaphin resistance was associated with mutations within the promoter region of the femAB operon. Four of the six characterized mutants contained an IS256 insertion within the femAB promoter and the remaining two mutants had an 18bp deletion within this region. IS256 insertion and the 18-bp deletions were associated with a reduction in femAB transcription and led to the production of an altered peptidoglycan muropeptide with interpeptide crossbridges consisting of a single glycine. The transcriptional start site of femAB was mapped to a 43-bp region of high homology between staphylococcal species, with five out of the six mutations occurring within this putative consensus region. We identified the femAB promoter region as the primary location of mutations associated with lysostaphin resistance.