To improve objectivity and speed of current antifungal mold susceptibility testing, the yeast Rapid Susceptibility Assay (RSA) was adapted for Aspergillus species. The RSA is based on glucose utilization in the presence of an antifungal drug. Aspergillus fumigatus conidia were incubated in 0.2% glucose RPMI 1640 containing 0.03 to 16 micro g of amphotericin B or itraconazole/ml. Drug-related inhibition of glucose utilization correlated with suppression of conidial germination. Following incubation of conidia with various concentrations of antifungal drug, the percentage of residual glucose in the growth medium was determined colorimetrically and plotted against drug concentration to determine the MIC (MIC(RSA)). National Committee for Clinical Laboratory Standards (NCCLS) M38-P testing was also performed to obtain NCCLS MICs (MIC(NCCLS)) for direct comparison with MIC(RSA)s. Conidial inocula of an optical density at 530 nm (OD(530)) of 0.11 facilitated determination of amphotericin B and itraconazole MIC(RSA)s at 16 h equal to or within a single twofold dilution of MIC(NCCLS)s obtained at 48 h. Preliminary testing with a 0.11-OD(530) conidial inoculum of the slower-growing Aspergillus terreus resulted in itraconazole and amphotericin B MIC(RSA)s at 16 h equal to or within a single twofold dilution of MIC(NCCLS)s obtained at 48 h. These data indicate that the mold RSA provides a more objective and rapid method for Aspergillus spp. susceptibility testing than the NCCLS M38-P assay.