OBJECTIVES: Trimethoprim/sulfamethoxazole (SXT) is used to treat Staphylococcus aureus infections. However, the effect of treatment with SXT is sometimes not sufficient and there are patients whose treatment has to be prolonged. There are few reports of isolated strains of SXT-resistant S. aureus, but it is possible that some resistant strains cannot be detected by current testing methods We have therefore developed a tool to identify these resistant strains. METHODS: The mutant selection window (MSW) of SXT for 40 clinical isolates of S. aureus, including 20 methicillin-resistant S. aureus (MRSA), was determined. The optimum concentration of SXT and thymidine in agar for detecting SXT-resistant small colony variants (SCVs) of S. aureus was investigated. RESULTS: The MSW50 and MSW90 of SXT, presented as a multiple of the minimum inhibitory concentration (MIC), were 16× MIC and >256× MIC, respectively. SCVs were detected within the MSW in 32 (80%) of the 40 clinical isolates studied. To maintain the morphology of SCVs, the most suitable concentrations of SXT and thymidine for screening were 4mg/L and 0.01μg/mL, respectively. All 32 SCVs were resistant to SXT (MIC >32mg/L). The sensitivity and specificity of this screening method was 100% and 88.9%, respectively. CONCLUSIONS: SXT-resistant SCVs are not usually detected by routine laboratory tests performed in hospitals. However, the screening test described here can easily distinguish SXT-resistant SCVs among S. aureus isolated from specimens. This newly developed screening test could become an important tool to prevent inappropriate use of SXT.