We studied the in vitro activity of voriconazole (VCZ) itraconazole (ITZ) and amphotericin B (AMB) against 216 clinical isolates of Aspergillus spp. (142 Aspergillus fumigatus and 74 nonfumigatus Aspergillus spp. isolates) using a broth macrodilution method. The MICs (microgram/mL) (mean, range) for A. fumigatus were: VCZ 0.88, 0.25-4; ITZ 0.54, 0.25-4; AMB 2.16, 0.5-8. MIC90s were: VCZ 2, ITZ 1, AMB 4. MICs for nonfumigatus Aspergillus spp. were: VCZ 1.57, 0.25-4; ITZ 1.74, 0.25-4; AMB 2.88, 0.5-8. MIC90s for this group were: VCZ 4, ITZ 4, AMB 4. We also studied the susceptibility to VCZ of 18 AMB-resistant (mean, MIC 6.0 micrograms/mL) and 28 ITZ-resistant (mean, MIC 13.28 micrograms/mL) A. fumigatus isolates selected in the laboratory. The mean MICs of VCZ were 0.59 microgram/mL for AMB-resistant and 1.32 micrograms/mL for ITZ-resistant isolates. Our study showed that VCZ and ITZ had comparable in vitro activity against the isolates studied, except against A. fumigatus, where the MIC of ITZ was lower. The azoles had better in vitro activity than AMB against A. fumigatus and non-fumigatus spp. The non-fumigatus Aspergillus spp. were less susceptible to all three antifungals evaluated. When tested against ITZ- or AMB-resistant A. fumigatus strains, VCZ retained good activity, showing only a modest rise in the MIC against ITZ-resistant strains.