Bupropion (1-(3-chlorophenyl)-2-((1,1-dimethylethyl) amino)-1-propanone) is a norepinephrine and dopamine reuptake inhibitor that is currently approved for use as an antidepressant (Wellbutrin), a smoking cessation aid (Zyban) and as a weight loss therapy in combination with naltrexone (Contrave). Bupropion is also the preferred sensitive in vivo CYP2B6 substrate recommended by the FDA. Yet bupropion is relatively insensitive to drug-drug interactions and its main elimination pathways in vivo in humans are still mainly uncharacterized. Bupropion is extensively metabolized, with less than 1% of the dose excreted unchanged in the urine. Bupropion has three primary active circulating metabolites; hydroxybupropion, threohydrobupropion, and erythrohydrobupropion but these metabolites and their conjugates represent less than 20% of the dose excreted in urine. The aim of this study was to characterize the remaining elimination pathways of bupropion and determine the relative contribution of CYP2B6 to overall bupropion clearance. Plasma and urine were analyzed from healthy subjects and three novel metabolites were identified and isolated. These novel metabolites and their conjugates were found to represent the majority of the dose excreted in urine. A hydroxylated product predominantly formed by CYP2C19 in vitro was found to constitute approximately 20% of the dose excreted. Sequential metabolites of erythro- and threohydrobupropion, also formed by CYP2C19 accounted for additional 20% of the dose. Taken together the data shows that formation of threo- and erythrohydrobupropion is the major elimination pathway for bupropion and that CYP2C19 plays an important role in overall bupropion clearance. These results suggest that observed inhibition of bupropion clearance by ticlopidine can be explained by CYP2C19 inhibition and is not a reliable measure of CYP2B6 activity. This study also shows that threohydrobupropion is the main primary elimination pathway of bupropion and therefore variation in 11B-HSD activity, will be a major contributor to variable pharmacokinetics of bupropion.