We investigated the effects of ketoconazole (KCZ) and fluconazole (FCZ) on rat liver microsomal nevirapine (NVP) metabolism in vitro and on NVP plasma profiles in vivo in order to determine whether the in vivo drug interactions could be predicted quantitatively from the in vitro data. The Ki values of KCZ and FCZ for NVP 12-hydroxylation were 1.59 microm and 11.5 microM, respectively, indicating that KCZ inhibited this activity more strongly than FCZ in vitro. In contrast, FCZ orally pre-administered at 20 mg/kg to rats increased the area under the plasma concentration-time curve (AUC) of NVP 7.4-fold, whereas KCZ increased it 2.1-fold, compared to the vehicle. We next investigated the inhibitory potency and unbound concentrations of KCZ and FCZ in microsomal mixtures with or without rat albumin. In the presence of albumin, the inhibition by KCZ was greatly decreased. Further, the unbound fraction of KCZ was decreased dramatically to around 3%, whereas more than 90% of FCZ remained in unbound form. When the increase in the AUC for NVP was calculated based on the concentrations of unbound inhibitors in the portal vein, good agreement with the observed in vivo values was obtained.