Candida glabrata is emerging as a more common and important human pathogen. It is less susceptible to azole antifungals than Candida albicans, thus, posing some unique treatment challenges. Previously undetected C. glabrata isolates were identified from clinical specimens by adding bile to the growth medium. Cholesterol was found to be the responsible ingredient in bile. Six bile-dependent isolates were characterized and were found to exhibit wild-type equivalent growth when provided human or bovine serum or free cholesterol. Sterol profiles of the 6 isolates and a C. glabrata matching wild-type strain not requiring cholesterol indicated that 2 were defective in squalene epoxidase (encoded by the ERG1 gene) activity, 3 were defective in lanosterol synthase (encoded by the ERG7 gene) activity, and the sixth was defective in heme biosynthesis. All 7 isolates produced profiles that contained cholesterol transported from the media. Because Saccharomyces cerevisiae mutants unable to synthesize heme will take up exogenous sterol under aerobic conditions, hem1 nulls of C. glabrata and C. albicans were generated and tested for growth on ergosterol media. Only the C. glabrata hem1 was able to grow indicating significant differences in exogenous sterol uptake between the 2 organisms. The ability of C. glabrata to replace ergosterol with host sterol may be responsible for its elevated azole resistance.