The impact of growth medium selection on antifungal susceptibility testing has been well documented. Previously we described the antifungal characteristics of LY 303366 via time-kill curve methods using RPMI 1640 buffered with 0.165 M morpholinepropane-sulfonic acid as growth medium. The purpose of the current study was to compare the previously reported kill curve results with results obtained using antibiotic medium number three (AM #3) as growth medium. Antifungal activity was assessed via susceptibility testing and time-kill studies in both media. Two isolates each of Candida albicans, C. glabrata, and C. tropicalis were studied. MICs for the six isolates were found to be 10 to 100 times lower in AM #3. Time-kill studies were conducted with multiples of the MIC ranging from 0.125 x MIC to 16 x MIC. LY 303366 exhibited fungicidal (> or = 3 log10 reduction in CFU) activity against all isolates in AM #3; however, fungicidal activity was noted only for three of the six isolates when tested in RPMI. Furthermore, the rate of fungicidal activity was more rapid when AM #3 was utilized. Not only were the rate and extent of activity influenced by choice of media, but the relationships between LY 303366 concentrations and activity were also found to be media dependent. The findings from this study serve to highlight further the importance of media selection for in vitro evaluation of antifungal activity. In vivo studies need to be conducted with LY 303366 to determine which media provides the best correlation between in vitro and in vivo findings.