The effect of itraconazole, a potent inhibitor of the CYP3A isoenzyme family, on the pharmacokinetics of imidafenacin, a novel synthetic muscarinic receptor antagonist, was investigated. Twelve healthy subjects participated in this open-label, self-controlled study. In period I, subjects received a single oral dose of 0.1 mg imidafenacin. In period II, they received multiple oral doses of 200 mg itraconazole for 9 days and a single oral dose of 0.1 mg imidafenacin on day 8. Plasma concentrations of imidafenacin and M-2, the major metabolite of imidafenacin metabolized by CYP3A4, were determined. Analytes were measured by liquid chromatography tandem mass spectrometry. Following coadministration with itraconazole, the maximum plasma concentration (C(max)) of imidafenacin increased 1.32-fold (90% confidence intervals [CIs]: 1.12-1.56), and the area under the plasma concentration-time curve from time 0 to infinity (AUC(0-infinity)) increased 1.78-fold (90% CI: 1.47-2.16). In conclusion, itraconazole increases the plasma concentrations of imidafenacin by inhibiting CYP3A4. Therefore, itraconazole or potent CYP3A4 inhibitors should be carefully added to imidafenacin drug regimens.