To investigate the in vitro activity of LY303366 (LY) against Candida isolates comprising nine different species and comparison with fluconazole (FLU), flucytosine (5FC) and amphotericin B (AMB). The method used was a microtitre modification of the NCCLS M27-A accepted standard using either RPMI-1640 with 2% glucose (5FC and FLU) or antibiotic medium 3 with 2% glucose (LY and AMB). The minimum inhibitory concentration (MIC) was the lowest drug concentration that reduced growth by 80% compared with the drug-free control. Minimum fungicidal concentrations (MFCs; 99% kill) were also determined for all isolates for LY and AMB. Overall, 58 of 105 (55.2%) isolates were resistant to FLU (MIC < or = 16 mg/L). There was no relationship between FLU and LY MICs for C. albicans or non-albicans species. For all isolates, geometric mean (GM) MIC values and ranges (in mg/L) were: LY 0.011 and < or = 0.001-16, FLU 8.72 and < or = 0.125- > 128, 5FC 0.393 and < or = 0.03- > 32, AMB 0.046 and 0.008-0.125. Differences in susceptibility to LY were seen: C. parapsilosis (n = 12, GM 0.4 and range 0.125-16) and C. guilliermondii (n = 8, GM 0.46 and range 0.25-1) were both found to be significantly less susceptible to LY than all other species (P < or = 0.05). For all isolates, geometric mean MFC values and ranges (in mg/L) were: LY 0.032 and 0.002-16, AMB 0.143 and 0.03-2. The MFC value was the same as or only one drug dilution higher than the MIC value for 69.5% and 48.6% of isolates tested for LY and AMB, respectively. Tolerance was described in 13.3% and 5.7% of isolates for LY and AMB, respectively. A reproducibility study performed on 20% of the isolates showed that 90.5%, 100%, 95.2% and 100% of isolates retested were the same or within one well of the original MIC value for LY, FLU, 5FC and AMB, respectively. LY303366 shows promising antifungal activity in vitro and warrants further in vivo investigation.