Mupirocin is a topical antimicrobial used to decolonize patients who carry methicillin-resistant Staphylococcus aureus (MRSA), and the topical agent retapamulin may be a potential alternative therapy. The goal of this study was to determine the in vitro activity of retapamulin as well as a panel of 15 antimicrobial agents, including mupirocin, for 403 MRSA isolates collected longitudinally from a naive population at the Veterans Affairs Puget Sound Health Care System. The MICs for retapamulin had a unimodal distribution, ranging from 0.008 to 0.5 μg/ml. One isolate had an MIC of >16 μg/ml, was also resistant to clindamycin and erythromycin, and was recovered from the nares of a patient undergoing hemodialysis. Twenty-four isolates (6%) and 11 isolates (3%) demonstrated low-level resistance (MICs of 8 to 64 μg/ml) and high-level resistance (MICs of ≥ 512 μg/ml), respectively, to mupirocin. Isolates were recovered from 10 patients both before and after mupirocin therapy. Of those, isolates from 2 patients demonstrated MIC changes postmupirocin therapy; in both cases, however, strain typing demonstrated that the pre- and postmupirocin strains were different. A total of 386 isolates (96%) had vancomycin MICs of ≤ 1.0 μg/ml; 340 isolates (84%) were resistant to levofloxacin, 18 isolates (4.5%) were resistant to trimethoprim-sulfamethoxazole, and 135 isolates (33%) had elevated MICs of 4 μg/ml for linezolid. The baseline levels of resistance were low for mupirocin (9%) and even lower for retapamulin (0.25%) Although the use of mupirocin is currently the standard therapy for decolonization practices, the activity of retapamulin warrants its consideration as an alternative therapy in MRSA decolonization regimens.