Candida dubliniensis is a recently described species of Candida associated with oral candidiasis in human immunodeficiency virus (HIV)-infected individuals. Nineteen oral isolates of C. dubliniensis recovered from 10 HIV-positive and 4 HIV-negative individuals and one vaginal isolate from an additional HIV-negative subject were assessed for fluconazole susceptibility by broth microdilution (BMD), hyphal elongation assessment, and Etest. The susceptibilities of these 20 isolates to itraconazole and amphotericin B and of 10 isolates to ketoconazole were also determined by BMD only. Sixteen of the C. dubliniensis isolates were susceptible to fluconazole (MIC range, 0.125 to 1.0 microgram ml-1), and four (recovered from two AIDS patients) were fluconazole resistant (MIC range, 8 to 32 micrograms ml-1). Fluconazole susceptibility data obtained by hyphal elongation assessment correlated well with results obtained by BMD, but the corresponding Etest MIC results were one to four times higher. All of the isolates tested were found to be sensitive to itraconazole, ketoconazole, and amphotericin B. Sequential exposure of two fluconazole-sensitive (MIC, 0.5 microgram ml-1) C. dubliniensis isolates to increasing concentrations of fluconazole in agar medium resulted in the recovery of derivatives which expressed a stable fluconazole-resistant phenotype (BMD-determined MIC range, 16 to 64 micrograms ml-1), even after a minimum of 10 consecutive subcultures on drug-free medium and following prolonged storage at -70 degrees C. The clonal relationship between the parental isolates and their respective fluconazole-resistant derivatives was confirmed by genomic DNA fingerprinting and karyotype analysis. The results of this study demonstrate that C. dubliniensis is inherently susceptible to commonly used antifungal drugs, that fluconazole resistance does occur in clinical isolates, and that stable fluconazole resistance can be readily induced in vitro following exposure to the drug.