The azole antifungals block ergosterol biosynthesis by inhibiting lanosterol demethylase (Erg11p). The resulting depletion of cellular ergosterol and the accumulation of "toxic" sterol intermediates are both thought to compromise plasma membrane function. However, the effects of ergosterol depletion upon the function of intracellular membranes and organelles are not well described. The purpose of this study was to characterize the effects of azole treatment upon the integrity of the Candida albicans vacuole and to determine whether, in turn, vacuolar trafficking influences azole susceptibility. Profound fragmentation of the C. albicans vacuole can be observed as an early consequence of azole treatment, and it precedes significant growth inhibition. In addition, a C. albicans vps21Δ/Δ mutant, blocked in membrane trafficking through the late endosomal prevacuolar compartment (PVC), is able to grow significantly more than the wild type in the presence of several azole antifungals under standard susceptibility testing conditions. Furthermore, the vps21Δ/Δ mutant is able to grow despite the depletion of cellular ergosterol. This phenotype resembles an exaggerated form of "trailing growth" that has been described for some clinical isolates. In contrast, the vps21Δ/Δ mutant is hypersensitive to drugs that block alternate steps in ergosterol biosynthesis. On the basis of these results, we propose that endosomal trafficking defects may lead to the cellular "redistribution" of the sterol intermediates that accumulate following inhibition of ergosterol biosynthesis. Furthermore, the destination of these intermediates, or the precise cellular compartments in which they accumulate, may be an important determinant of their toxicity and thus ultimately antifungal efficacy.