The influence of test variables on in vitro susceptibility testing of caspofungin was examined with 694 isolates of Candida albicans including seven laboratory-derived glucan synthesis mutants. The conditions providing the greatest separation between the mutant strains and the clinical isolates were RPMI medium, MIC end point criterion of partial inhibition, and incubation for 24 h. These testing conditions were then applied to 3,322 isolates of Candida spp. (3,314 clinical isolates and eight glucan synthesis mutants). Among the 11 isolates for which caspofungin MICs were >/=2 microg/ml, eight were accounted for by the glucan synthesis mutants. The MICs for >99% of isolates were </=1 microg/ml, and thus these isolates were differentiated from strains with reduced in vitro and in vivo susceptibilities to caspofungin.