Prasugrel and clopidogrel inhibit platelet aggregation through active metabolite formation. Prasugrel's active metabolite (R‐138727) is formed primarily by cytochrome P450 (CYP) 3A and CYP2B6, with roles for CYP2C9 and CYP2C19. Clopidogrel's activation involves two sequential steps by CYP3A, CYP1A2, CYP2C9, CYP2C19, and/or CYP2B6. In a randomized crossover study, healthy subjects received a loading dose (LD) of prasugrel (60 mg) or clopidogrel (300 mg), followed by five daily maintenance doses (MDs) (15 and 75 mg, respectively) with or without the potent CYP3A inhibitor ketoconazole (400 mg/day). Subjects had a 2‐week washout between periods. Ketoconazole decreased R‐138727 and clopidogrel active metabolite Cmax (maximum plasma concentration) 34–61% after prasugrel and clopidogrel dosing. Ketoconazole did not affect R‐138727 exposure or prasugrel's inhibition of platelet aggregation (IPA). Ketoconazole decreased clopidogrel's active metabolite AUC0–24 (area under the concentration–time curve to 24 h postdose) 22% (LD) to 29% (MD) and reduced IPA 28% (LD) to 33% (MD). We conclude that CYP3A4 and CYP3A5 inhibition by ketoconazole affects formation of clopidogrel's but not prasugrel's active metabolite. The decreased formation of clopidogrel's active metabolite is associated with reduced IPA. Clinical Pharmacology & Therapeutics (2007) 81, 735–741. doi:10.1038/sj.clpt.6100139; published online 14 March 2007