The prodrug clopidogrel (Plavix) is activated by cytochrome P450 (P450) to a metabolite that inhibits ADP-induced platelet aggregation. Clopidogrel is frequently administered to patients in conjunction with the CYP3A4 substrate atorvastatin (Lipitor). Since clinical studies indicate that atorvastatin inhibits the antiplatelet activity of clopidogrel, we investigated whether CYP3A4 metabolized clopidogrel in vitro. Microsomes prepared from dexamethasone-pretreated rats metabolized clopidogrel at a rate of 3.8 nmol min-1nmol of P450-1, which is 65 and 1270% faster than the rate of metabolism by microsomes from control and ß-napthoflavone-treated rats, respectively. To identify the human P450s responsible for clopidogrel oxidation, genetically engineered microsomes containing a single human P450 isozyme were tested for their ability to oxidize clopidogrel. CYP3A4 and 3A5 metabolized clopidogrel at a significantly higher rate than eight other P450 isozymes, suggesting that CYP3A4 and 3A5 are primarily responsible for in vivo clopidogrel metabolism. Clopidogrel interacts with human CYP3A4 with a spectral dissociation constant (Ks),Km, and Vmax of 12 µM, 14 ± 1 µM and 6.7 ± 1 nmol min-1nmol P450-1, respectively. Atorvastatin lactone, the physiologically relevant substrate, inhibits clopidogrel with aKi of 6 µM. When clopidogrel and atorvastatin are present at equimolar concentrations, clopidogrel metabolism is inhibited by greater than 90%. Since CYP3A4 and 3A5 metabolize clopidogrel faster than other human P450 isozymes and are the most abundant P450s in human liver, they are predicted to be predominantly responsible for the activation of clopidogrel in vivo.