Prior to characterization of antifungal inhibitors that target CYP51, CYP51 was expressed in , purified, and characterized. CYP51 bound lanosterol, obtusifoliol, and eburicol with similar affinities (dissociation constant [ ] values, 22.7, 20.3, and 20.9 μM, respectively) but displayed substrate specificity, insofar as only eburicol was demethylated in CYP51 reconstitution assays (turnover number, 1.55 min; value, 2 μM). The investigational agent VT-1161 bound tightly to CYP51 ( = 242 nM) with an affinity similar to that of clotrimazole, fluconazole, ketoconazole, and voriconazole ( values, 179, 173, 312, and 304 nM, respectively) and with an affinity lower than that of itraconazole ( = 53 nM). Determinations of 50% inhibitory concentrations (ICs) using 0.5 μM CYP51 showed that VT-1161 was a tight-binding inhibitor of CYP51 activity, yielding an IC of 0.14 μM, whereas itraconazole, fluconazole, and ketoconazole had ICs of 0.26, 0.4, and 0.6 μM, respectively. When the activity of VT-1161 was tested against 34 clinical isolates, VT-1161 was a potent inhibitor of growth, with MIC, MIC, and geometric mean MIC values of ≤0.03, 0.06, and 0.033 μg ml, respectively. With its selectivity versus human CYP51 and drug-metabolizing cytochrome P450s having already been established, VT-1161 should prove to be safe and effective in combating infections in patients.