1. Four enzymes were purified to homogeneity from human liver cytosol and were demonstrated to be responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). 2. Carbonyl reductase (EC 18.104.22.168), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, was compared with three isoenzymes of the aldo-keto reductase (AKR) superfamily in terms of enzyme kinetics, co-substrate dependence and inhibition pattern. 3. AKR1C1, 1C2 and 1C4, previously designated as dihydrodiol dehydrogenases (DD1, DD2 and DD4), showed lower K(m) (0.2, 0.3 and 0.8 mM respectively) than did carbonyl reductase (7 mM), whereas carbonyl reductase exhibited the highest enzyme efficiency (Vmax/K(m)) for NNK. Multiplication of enzyme efficiencies with the relative quantities of individual enzymes in cytosol resulted in a rough estimate of their contributions to total alcohol metabolite formation. These were approximately 60% for carbonyl reductase, 20% each for AKR1C1 and 1C2, and 1% for AKR1C4. 4. Except for AKR1C4, the enzymes had a strong preference for NADPH over NADH, and the highest activities were measured with an NADPH-regenerating system. Carbonyl reductase activity was extensively inhibited by menadione, rutin and quercitrin, whereas medroxyprogesterone acetate, phenolphthalein and flufenamic acid were potent inhibitors of AKR1C1, 1C2 and 1C4. 5. In conclusion, cytosolic members of the SDR and AKR superfamilies contribute to reductive NNK detoxification in human liver, the enzymes responsible being carbonyl reductase and aldoketo reductases of the AKRIC subfamily.