The methyltransferase genes erm(B) and cfr are adjacent to each other in the chromosome of methicillin-resistant Staphylococcus aureus strain CM05. Analyses of the transcriptional organization of the erm(B) and cfr genes in the chromosome of strain CM05 showed that the two genes are organized into an operon, designated mlr (for modification of the large ribosomal subunit), which is controlled by the erm(B) promoter. Analysis of the translation control and the inducibility of the erm(B) and cfr genes in the mlr operon showed that despite the presence of putative regulatory short open reading frames, both genes are expressed constitutively. The combined action of the two methyltransferases encoded in the mlr operon results in modification of two specific residues in 23S rRNA, A2058 and A2503, and renders cells resistant to all clinically useful antibiotics that target the large ribosomal subunit. Furthermore, simultaneous modification of both rRNA sites synergistically enhances resistance to 16-member-ring macrolides.