The irreversible inhibition of cytochrome P450 (CYP) enzymes can cause significant drug-drug interactions (DDIs). The formation of metabolites is fundamental for the inactivation of CYP enzymes. Of the 19 CYP enzyme inactivators for which the mechanism of action has been established, 10 have circulating metabolites, which are on the metabolic pathway to inactivation of the CYP enzyme. Because inactivation of CYP enzymes usually requires multiple metabolic steps, the prediction of interactions between metabolites and CYPs in vivo may require complex models and the availability of data generated in vitro from each metabolite. Data discussed in this review suggest that circulating metabolites are more important in CYP inhibition in vivo than has been acknowledged.