Bupropion is a widely used antidepressant drug and also as a smoking cessation aid. In humans, this drug is extensively metabolised to form three major metabolites: oxidised hydroxybupropion and two reduced metabolites threohydrobupropion and erythrohydrobupropion. All of these metabolites are considered to be active. Although the oxidative metabolic pathway and the central role of CYP2B6 are known, the enzymes that participate in the reduction have not been identified to date. The aim of this study was to elucidate the contribution of the reduction reaction to the bupropion metabolism and to confirm the role of human liver subcellular fractions in the metabolism. An optimised HPLC method for the determination of bupropion metabolites was utilised. The recombinant forms of ten well-known carbonyl-reducing enzymes were tested to determine activities in the reduction of bupropion. This study provides the first direct evidence that five of the ten tested enzymes participate in the reducing biotransformation of bupropion in vitro. The enzyme kinetics of all of these reductases were investigated and kinetic parameters were calculated. The in vitro results indicated that 11β-hydroxysteroid dehydrogenase 1 is responsible for the formation of the highest amount of threohydrobupropion whereas AKR1C1 exhibits the highest efficiency in the production of erythrohydrobupropion.