The usefulness of a colorimetric method using Alamar Blue (Alamar Blue method) for susceptibility testing of Aspergillus species to voriconazole and three existing antifungal agents, itraconazole (ITCZ), flucytosine (5-FC), and amphotericin B (AMPH-B), was studied using four ATCC strains of three species, including two strains of A. fumigatus and one each of A. flavus and A. niger. For comparison, the broth microdilution method for antifungal susceptibility testing of filamentous fungi proposed by the National Committee for Clinical Laboratory Standards (NCCLS method M38-P) was also used. Minimum inhibitory concentration (MIC) endpoints were read spectrophotometrically for the Alamar Blue method and visually for the NCCLS method after 46-50 h. Like the NCCLS method, Alamar Blue produced highly reproducible results for all the drugs and strains tested; most MIC values obtained by nine tests were within the range of 2 twofold dilutions for each strain. Voriconazole and ITCZ susceptibility testing with the Alamar Blue method and the NCCLS method yielded comparable results in 94% of the tests, meaning that the endpoints obtained were identical or differed by no more than 2 twofold dilutions. On the other hand, susceptibility testing for 5-FC and AMPH-B yielded scores of 25% and 64%, respectively. Our study suggests the potential value of the Alamar Blue method as a convenient alternative to the NCCLS M38-P method for routine testing of Aspergillus species susceptibility to at least voriconazole and ITCZ.