The allosteric effect of fluconazole (effector) on the formation of 1'-hydroxymidazolam (1'-OH-MDZ) and 4-hydroxymidazolam (4-OH-MDZ) from midazolam (MDZ), a substrate of CYP3A4/5—members of the cytochrome P450 superfamily of enzymes—was examined in healthy volunteers. Following pretreatment with fluconazole, the ratio of the areas under the curve (AUCs) for 4-OH-MDZ and MDZ (AUC4-OH/AUCMDZ) increased by 35-62%, whereas the ratio AUC1'-OH/AUCMDZ decreased by 5-37%; the ratio AUC1'-OH/AUC4-OH decreased by 46-58% after fluconazole administration and had no association with theCYP3A5genotype. Thein vitroformation of 1'-OH-MDZ was more susceptible to inhibition by fluconazole than that of 4-OH-MDZ. Fluconazole decreased the intrinsic formation-clearance ratio of 1'-OH-MDZ/4-OH-MDZ to an extent that was quantitatively comparable toin vivoobservations. The elimination clearance of MDZ metabolites appeared unaffected by fluconazole. This study demonstrated that fluconazole alters formation of MDZ metabolites, bothin vivoandin vitro,in a manner consistent with an allosteric interaction. The 1'-OH-MDZ/4-OH-MDZ ratio may serve as a biomarker of such interactions among MDZ, CYP3A4/5, and other putative effectors.