We have reported that the gene mph(C) (formally referred to as 'mphBM') is located on plasmid pMS97 342 bp downstream of the msr(A) gene. msr(A) specifies resistance to macrolides by ABC-transporter-mediated efflux, and mph(C) has 49% identity to the amino acid sequence of MPH(2')II, which encodes a phosphotransferase that inactivates some macrolide antibiotics. A strain of Staphylococcus aureus NCTC8325 containing plasmid pMS97 inactivated unlabeled and (14)C-labeled erythromycin when tested by bioautographic and radioautographic techniques. In addition to erythromycin, other 14-membered ring macrolides (except for ketolides), 15-membered ring macrolides and 16-membered ring macrolides, mycinamicin, rosamicin and YM133, were inactivated by the strain. Erythromycin inactivation products produced by the strain carrying pMS97 were completely different from those produced by Escherichia coli BM694 bearing plasmid pAT63, which contains the ereA gene encoding an esterase that hydrolyzes macrolide lactones. Constructs formed with the msr(A) and mph(C) genes, and with the msr(A), mph(C) and erm(Y) genes, showed erythromycin-inactivating activity, but another construct built with the mph(C) gene alone failed to show such activity. This result suggests that any region of the msr(A) gene is needed for the expression of mph(C).