The susceptibilities to mupirocin of 102 selected clinical isolates of Staphylococcus aureus and of control strain S. aureus NCTC 6571 were determined by disc diffusion (using discs containing 5, 15, 25, 30, 50 and 200 microg of mupirocin) and Etest and the results were compared with MICs determined using an agar incorporation method. On the basis of agar incorporation MICs, 42 isolates were sensitive to mupirocin (MIC < or = 4 mg/L), 39 showed low-level resistance (MICs = 8-128 mg/L) and 22 were highly resistant (MICs > or = 256 mg/L) and contained the mupA resistance gene. Using Stokes' criteria, none of the discs used gave major errors (sensitive isolates classified as highly resistant) or very major errors (highly resistant isolates classified as sensitive) in assigning a category of susceptibility, but minor errors (a difference of one category) were noted with all strengths. The best correlation with agar incorporation MIC was obtained with 25 microg mupirocin discs, which classified correctly 98 (95%) isolates, while worse correlations were noted with 5 microg and 200 microg discs which are the only types currently available commercially, for which there were 47 and 30 minor errors, respectively. The MICs found by Etest were the same as, or lower than, those determined by agar incorporation. Etests classified correctly all 42 mupirocin-sensitive isolates, 19 (49%) low-level resistant isolates and 16 (73%) highly resistant isolates. Two isolates that contained the mupA gene and showed agar incorporation MICs of 256 mg/L and 512 mg/L were not classified as highly resistant by any of the diffusion methods used. Agar incorporation MIC determination, possibly supported by detection of the mupA gene, offers the most effective means of identifying high-level mupirocin resistance in S. aureus, although the Etest also proved to be reproducible. However, we conclude that 25 microg discs warrant further evaluation for possible use in clinical laboratories, as they appear to be more reliable than the discs currently available.