A comparative evaluation of the in vitro susceptibilities of 597 clinical yeast isolates to amphotericin B, fluconazole, and 5-fluorocytosine (5FC) was conducted. The broth macrodilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS, M27-P) was adapted to the microdilution method. Microdilution endpoints for amphotericin B were scored as the lowest concentration in which a score of 0 (complete absence of growth) was observed and for 5FC and fluconazole as the lowest concentration in which a score of 2 (prominent decrease in turbidity; MIC-2) was observed compared to the growth control. The MIC values were read after 24 and 48 h incubation. A broad range of MIC values was observed with each antifungal agent. Amphotericin B was very active (MIC90 < or = 1.0 microgram/ml) against all of the yeast isolates with the exception of C. lusitaniae (MIC90 > or = 2.0 micrograms/ml). Fluconazole was most active against C. parapsilosis (MIC90 of 1.0 microgram/ml) and least active against C. krusei (MIC90 of 32 micrograms/ml). 5FC was most active against C. albicans, C. parapsilosis, C. tropicalis, and T. glabrata (MIC90 < or = 1.0 microgram/ml) and was least active against C. krusei and C. lusitaniae (MIC90 > or = 16 micrograms/ml). These data indicate that the microdilution method, performed in accordance with M27-P, provides a means of testing larger numbers of yeast isolates against an array of antifungal agents and allows this to be accomplished in a reproducible and standardized manner. Given these results, it appears that the microdilution method may be a useful alternative to the macrodilution reference method for susceptibility testing of yeasts.