Bupropion hydroxylation is a bioactivation and metabolic pathway, and the standard clinical CYP2B6 probe. This investigation determined the influence ofCYP2B6allelic variants on clinical concentrations and metabolism of bupropion enantiomers. Secondary objectives evaluated the influence ofCYP2C19and P450 oxidoreductase variants. Healthy volunteers in specific cohorts (CYP2B6*1/*1, CYP2B6*1/*6, CYP2B6*6/*6, and alsoCYP2B6*4carriers) received single‐dose oral bupropion. Plasma and urine bupropion and hydroxybupropion was quantified. Subjects were also genotyped forCYP2C19and P450 oxidoreductase variants. Hydroxylation of both bupropion enantiomers, assessed by plasma hydroxybupropion/bupropion AUC ratios and urine hydroxybupropion formation clearances, was lower inCYP2B6*6/*6but notCYP2B6*1/*6compared withCYP2B6*1/*1genotypes, and numerically greater inCYP2B6*4carriers.CYP2C19and P450 oxidoreductase variants did not influence bupropion enantiomers hydroxylation or plasma concentrations. The results show that clinical hydroxylation of both bupropion enantiomers was equivalently influenced byCYP2B6allelic variation.CYP2B6polymorphisms affect S‐bupropion bioactivation, which may affect therapeutic outcomes.