OBJECTIVE To examine the in vitro activities of five antifungal agents against common pathogenic Exophiala species; to detect the minimum inhibitory concentration (MIC) discrepancies among different drugs and different species; to evaluate the role of the influence factors in MIC determination; and to establish a standard method for the antifungal susceptibility testing of Exophiala spp. METHODS MICs of itraconazole (ICZ), fluconazole, ketoconazole, amphotericin B (AmB) and 5-flucytosine against 5 species (57 strains) of Exophiala spp were determined by modified NCCLS M27-A broth microdilution method. Two inoculum sizes (0.5 x 10(3)-2.5 x 10(3) CFU/ml and 0.5 x 10(4)-2.5 x 10(4) CFU/ml) and three incubation times (4, 7 and 10 days) were evaluated. The minimum fungicidal concentrations (MFC) of itraconazole against 20 Exophiala strains were also detected. RESULTS All the tested Exophiala species were sensitive to AmB and ICZ. MICs varied among different species and drugs. Resistant strains to different drugs existed in this genus. The proper inoculum density was about 0.5 x 10(3) CFU/ml to 2.5 x 10(3) CFU/ml, and the 4-day incubation time was suitable for more than 90% of the strains. CONCLUSIONS AmB or ICZ may serve as the first choice in treatment of phaeohyphomycosis. A standard microdilution procedure for MIC detection of Exophiala species is established in this experiment. The inoculum size and incubation time are the essential factors in the standardization of antifungal susceptibility testing of filamentous fungi. We hope this experiment may correlate well with the clinical treatment of phaeohyphomycosis caused by Exophiala species.