Candida krusei has become an increasingly important invasive pathogen, particularly in AIDS patients and is highly resistant to fluconazole. In vitro growth inhibition studies revealed that fluconazole and ketoconazole were approximately 800- and 19-fold less inhibitory than itraconazole. The inhibition and interaction of itraconazole, fluconazole and ketoconazole with the sterol 14 alpha-demethylase of C. krusei was studied using in vitro ergosterol biosynthesis and difference spectroscopy, respectively. Both itraconazole and ketoconazole inhibited in vitro ergosterol biosynthesis at lower concentrations than fluconazole. All three drugs interacted with microsomal P450 and interfered in the binding of carbon monoxide to P450 in direct proportion to their inhibitory effect on ergosterol biosynthesis in cell-free extracts. The slightly lower affinity of sterol 14 alpha-demethylase for fluconazole compared with itraconazole and ketoconazole is only partially responsible for poor activity of fluconazole on C. krusei.