The in vitro activities of amphotericin B, itraconazole, and voriconazole against 283 clinical isolates of Aspergillus fumigatus were studied by comparing the E test method with the reference procedure NCCLS (CLSI) M-38A. The methods were considered to agree when the results of the MICs by E test were within +/-2 dilutions of the MICs obtained by CLSI M-38 A. Agreement of readings at 24, 48, and 72 h of incubation was as follows: amphotericin B: 4.2%, 98%, and 40.6%; itraconazole: 92.5%, 100%, and 89.9%; and voriconazole: 98.9%, 100%, and 99.7%. The correlation between methods to classify the strains as "susceptible" or "resistant" was very good for itraconazole and voriconazole after 48 h of incubation, but 23% of the strains were incorrectly classified by E test for amphotericin B. The E test is an efficacious method for antifungal susceptibility testing in A. fumigatus for itraconazole and voriconazole when the plates are read after 48 h of incubation. The use of the E test to study the activity of amphotericin B should be avoided.