ABSTRACT We have previously reported poor correlation between the in vitro fungicidal activity of LY303366 and MIC results in RPMI medium based upon the manufacturer’s suggested susceptibility endpoint, lack of visual growth. Additionally, we have noted a significant trailing effect with LY303366 when MICs are determined in RPMI medium. These observations have led us to evaluate an alternative susceptibility endpoint for LY303366, an 80% reduction in growth compared with control (similar to that utilized for azoles). Two isolates each of Candida albicans , Candida glabrata , and Candida tropicalis were selected for testing. MICs were determined for LY303366 in RPMI 1640 medium buffered with morpholinepropanesulfonic acid. MICs were determined with suggested (MIC 100 ) and experimental (MIC 80 ) endpoints. The minimal fungicidal concentration (MFC) of LY303366 for each isolate was also determined. Time-kill curves were determined in RPMI medium with each isolate at concentrations of LY303366 ranging from 0.125 to 16× MIC 80 to assess the correlation between MIC 80 and fungicidal activity. Lastly, fungi exposed to LY303366 were examined via scanning electron microscope (SEM) for evidence of drug-induced ultrastructure change. MIC 80 s for test isolates ranged from 0.015 to 0.12 μg/ml and were consistently three to five wells less than MIC 100 s. Good correlation was observed between fungicidal activity, as assessed by kill curves, and the MIC 80 . SEM data revealed significant ultrastructure changes induced by LY303366 even at sub-MIC 80 s. Based on our results demonstrating better correlation between MIC 80 and fungicidal activity, i.e., time-kill curves and MFCs, we suggest that 80% reduction in visible growth be utilized as the endpoint for susceptibility determinations with LY303366 in RPMI medium.