The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen, independent of the route and type of administration. There are competing metabolic activation and detoxification pathways. NNK is activated by alpha-hydroxylation at either the methyl or methylene carbonyl adjacent to the N-nitroso group to yield intermediates that methylate and pyridyloxobutylate DNA. Detoxification of NNK in humans usually occurs via carbonyl reduction to its hydroxy product NNAL, which undergoes glucuronosylation and final excretion. In vitro studies on NNK metabolism have usually been performed with tissue homogenates, microsomal fractions and/or purified microsomal enzymes, but cytosolic metabolism of NNK has been ignored until today. The results of this study demonstrate that cytosolic fractions of human liver and lung also participate in NNK metabolism. We provide evidence that a substantial degree of NNK carbonyl reduction occurs by cytosolic enzymes and that these enzymes may contribute to NNK detoxification in human liver and lung. The relative contribution of cytosolic vs. microsomal NNK carbonyl reduction is nearly identical in liver, whereas it is more than 3-fold higher in lung microsomes compared to lung cytosol. The inhibition profile suggested that mainly carbonyl reductase (EC 1.1.1.184) was active in cytosol of both organs. The expression of carbonyl reductase mRNA in liver and lung was proven by reverse transcription-(RT)-PCR. In conclusion, the results of this study provide the first data on cytosolic enzymes participating in NNK detoxification in human liver and lung.