The clearance of racemic bupropion, metabolized selectively by CYP2B6 in vitro, has been used clinically to phenotype CYP2B6 activity, polymorphisms, and drug interactions but has known limitations. Bupropion hydroxylation by CYP2B6 is stereoselective. This investigation assessed the stereoselectivity of bupropion pharmacokinetics and the influence of CYP2B6 induction. Ten healthy volunteers received immediate-release bupropion before and after 7 days of rifampin. Plasma and urine bupropion and hydroxybupropion were analyzed using a stereoselective assay. Plasma area under the curve (AUC(0-infinity)) and maximum concentrations were 3-fold greater for R- than S-bupropion. Bupropion apparent oral clearance was 3- and 2-fold greater for S- than R- and R,S-bupropion, respectively. Hydroxybupropion plasma AUC(0-infinity) and elimination half-life were significantly less for (S,S)- than (R,R)- and the racemate. (S,S)-hydroxybupropion was formation rate limited, whereas (R,R)-hydroxybupropion and the racemate were elimination rate limited. Rifampin doubled both R- and S-bupropion clearance and caused 4-fold increases in both (R,R)- and (S,S)-hydroxybupropion formation clearances. Increases in the plasma hydroxybupropion/bupropion AUC(0-infinity) ratio were greater for (S,S)- than (R,R)-hydroxybupropion. Simplified plasma and urine metrics of stereoselective bupropion metabolism and clearance were identified. Because metabolite formation clearance is the best in vivo metric of enzyme activity and due, therefore, to faster S-bupropion elimination and formation rate-limited (S,S)-hydroxybupropion kinetics, stereoselective S-bupropion hydroxylation and (S,S)-hydroxybupropion formation clearance may be a useful and improved phenotypic probe for CYP2B6.