OBJECTIVE Telaprevir inhibits CYP3A resulting in drug-drug interactions (DDI) of unprecedented magnitude. We investigated the mechanisms by which telaprevir inhibits the oxidation of midazolam and tacrolimus in human liver microsomes (HLM). METHODS We performed a static mechanistic DDI prediction to evaluate whether previously reported competitive inhibition of CYP3A by telaprevir and its diastereomeric metabolite - VRT-127394 is sufficient to explain the remarkable reduction in oral clearance observed with oral midazolam and tacrolimus. To further explore the inhibitory mechanisms of telaprevir, we assessed whether telaprevir-mediated inhibition of the oxidation of midazolam and tacrolimus is time-dependent in human liver microsomes, and whether any observed time-dependency was irreversible or reversible in nature. RESULTS The competitive inhibition model failed to account for the magnitude of telaprevir interactions in human subjects. In comparing HLM incubations with and without a prior 30-min exposure to telaprevir, a respective 4- and 11-fold reduction in IC50 was observed with midazolam and tacrolimus as substrates. This time-dependent inhibition was shown to be NADPH-dependent. Upon dilution of microsomes following pre-incubation with telaprevir, time-dependent inhibition of midazolam metabolism was completely reversed, whereas partial reversal occurred with tacrolimus. CONCLUSIONS The interaction between telaprevir and midazolam or tacrolimus involves both competitive and time-dependent inhibition. The time-dependent component is not explained by irreversible inactivation of CYP3A. Formation of potent inhibitory metabolites may contribute to the remarkable in vivo inhibitory potency of telaprevir.