Ambrisentan is an endothelin type A (ETA)-selective receptor antagonist that is metabolized primarily by glucuronidation but also undergoes oxidative metabolism by CYP3A4. The potential for ketoconazole, the archetypal strong inhibitor of CYP3A4, to alter the pharmacokinetic profile of ambrisentan and its oxidative metabolite, 4-hydroxymethyl ambrisentan, was assessed in an open-label, nonrandomized, 2-period, single-sequence study in 16 healthy men. Participants received a single dose of ambrisentan 10 mg alone and after 4 days of ketoconazole 400 mg administered once daily. In the presence of multiple doses of ketoconazole, single-dose ambrisentan AUC0-∞ estimate was increased by 35.3%, whereas Cmax was increased by 20.0%. For the 4-hydroxymethyl ambrisentan metabolite, AUC0-∞ estimate was decreased by 4.0%, whereas Cmax was decreased by 16.5%. Concomitant administration of ambrisentan and ketoconazole was well tolerated. In summary, ketoconazole had no clinically significant effect on the pharmacokinetics or safety profile of ambrisentan; therefore, no changes in ambrisentan dose should be necessary when the drug is administered concomitantly with known CYP3A4 inhibitors.