Mechanisms of resistance to azoles in Candida albicans, the main etiologic agent of oropharyngeal candidiasis (OPC), include alterations in the target enzyme (lanosterol demethylase) and increased efflux of drug. Previous studies on mechanisms of resistance have been limited by the fact that only a single isolate from each OPC episode was available for study. Multiple isolates from each OPC episode were evaluated with oral samples plated in CHROMagar Candida with and without fluconazole to maximize detection of resistant yeasts. A total of 101 isolates from each of three serial episodes of OPC from four different patients were evaluated. Decreasing geometric means of fluconazole MICs with serial episodes of infection were detected in the four patients. However, 8-fold or larger (up to 32-fold) differences in fluconazole MICs were detected within isolates recovered at the same time point in 7 of 12 episodes. Strain identity was analyzed by DNA typing techniques and indicated that isolates from each patient represented mainly isogenic strains, but differed among patients. A Northern blot technique was used to monitor expression of ERG11 (encoding lanosterol demethylase) and genes coding for efflux pumps. This analysis revealed that clinical isolates obtained from the same patient and episode were phenotypically heterogeneous in their patterns of expression of these genes involved in fluconazole resistance. These results demonstrate the complexity of the distribution of the molecular mechanisms of antifungal drug resistance and indicate that different subpopulations of yeasts may coexist at a given time in the same patient and may develop resistance through different mechanisms.