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With increasing pressure to prevent methicillin-resistant Staphylococcus aureus (MRSA) infection, it is possible that there will be increased use of mupirocin for nasal decolonization of MRSA. Understanding the mechanisms, clinical significance, and epidemiology of mupirocin resistance is important for predicting how changes in mupirocin use may affect bacterial populations and MRSA control. High-level mupirocin resistance in S. aureus is mediated by a plasmid-encoded mupA gene. This gene can be found on conjugative plasmids that carry multiple resistance determinants for other classes of antimicrobial agents. High-level resistance has been associated with decolonization failure, and increased resistance rates have been associated with increased mupirocin use. Low-level mupirocin resistance is mediated via mutation in the native ileS gene, and the clinical significance of this resistance is unclear. Laboratory tests to detect and distinguish between these types of resistance have been described but are not widely available in the United States. Institutions that are considering the implementation of widespread mupirocin use should consider these resistance issues and develop strategies to monitor the impact of mupirocin use.