Drug-drug interactions involving efavirenz are of major concern in clinical practice. We evaluated the effects of multiple doses of efavirenz on omeprazole 5-hydroxylation (CYP2C19) and sulfoxidation (CYP3A). Healthy volunteers (n = 57) were administered a single 20 mg oral dose of racemic omeprazole either with a single 600 mg oral dose of efavirenz or after 17 days of administration of 600 mg/day of efavirenz. The concentrations of racemic omeprazole, 5-hydroxyomeoprazole (and their enantiomers), and omeprazole sulfone in plasma were measured using a chiral liquid chromatography-tandem mass spectrometry method. Relative to single-dose treatment, multiple doses of efavirenz significantly decreased (P < 0.0001) the area under the plasma concentration-time curve from 0 to infinity (AUC(0-∞)) of racemic-, R- and S-omeprazole (2.01- to 2.15-fold) and the corresponding AUC(0-∞) metabolic ratio (MR) for 5-hydroxyomeprazole (1.36- to 1.44-fold) as well as the MR for omeprazole sulfone (∼2.0) (P < 0.0001). The significant reduction in the AUC of 5-hydroxyomeprazole after repeated efavirenz dosing suggests induction of sequential metabolism and mixed inductive/inhibitory effects of efavirenz on CYP2C19. In conclusion, efavirenz enhances omeprazole metabolism in a nonstereoselective manner through induction of CYP3A and CYP2C19 activity.