Itraconazole (ITZ) is a substrate of CYP3A and both ITZ and hydroxyitraconazole (OH-ITZ), a major metabolite formed by CYP3A, are potent inhibitors of CYP3A. The concentration- and time-dependent changes in the hepatic availability (F(H)) of ITZ were evaluated in rats after oral doses of 5 and 40 mg/kg. Simultaneous blood samples were obtained from the aorta, portal vein, and hepatic vein for 24 h following duodenal ITZ administration, and concentrations of ITZ and OH-ITZ determined by LC/MS. During the absorption phase, the F(H) of ITZ increased from 0.2 to 1.0, reflecting the time course of hepatic CYP3A inhibition. A counterclockwise hysteresis was observed between ITZ concentrations entering the liver (C(IN,ITZ)) and F(H), whereas there was no time delay observed between the change in F(H) and the OH-ITZ concentrations entering the liver (C(IN,OH-ITZ)). The direct relationship between C(IN,OH-ITZ) and F(H) suggested that OH-ITZ was mainly responsible for the inhibition of CYP3A. A positive portal venous-aortic gradient for OH-ITZ was measured after duodenal administration of ITZ, indicating intestinal formation of OH-ITZ. The in vivo Ki for OH-ITZ (38 +/- 3 nM) was estimated from C(IN,OH-ITZ) versus F(H) of ITZ, and is similar to values obtained from inhibition of midazolam hydroxylation in CYP3A4 supersomes (Drug Metab Dispos 32:1121-1131, 2004). The data suggest that OH-ITZ, formed by intestinal CYP3A, controls the time course of hepatic CYP3A inhibition and is mainly responsible for the observed increase in F(H) of ITZ.