Ritonavir, a potent inhibitor of cytochrome P450 isoform 3A (CYP3A) activity, is frequently used to boost the effects of protease inhibitors at doses of 100-400 mg per day; however, human data regarding the optimal dose required for boosting are limited. This study systematically evaluated the ritonavir dose-response relationship on presystemic and systemic CYP3A metabolism using the human immunodeficiency virus integrase inhibitor elvitegravir and midazolam as probe substrates. Ritonavir administered once daily with elvitegravir exhibited nonlinear pharmacokinetics, with a 119-fold increase in the area under the plasma concentration-time curve over the dosing interval over a 20- to 200-mg dose range. The 20-mg dose of ritonavir substantially reduced CYP3A-mediated clearance (CL), as evidenced by a 66% reduction in midazolam CL that plateaued to 17% of baseline activity at a 100-mg dose. Maximum inhibition of elvitegravir apparent oral CL was achieved with ritonavir doses of 50-100 mg. Elvitegravir and ritonavir were generally well tolerated in this study. These data provide a critical understanding of ritonavir's dose-response relationship for inhibition of CYP3A activity in humans.