The N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco and tobacco smoke. Carbonyl reduction, alpha-carbon hydroxylation (activation) and N-oxidation of the pyridyl ring (detoxification) are the three main pathways of metabolism of NNK. In this study, metabolism of NNK was studied with lung and liver microsomes from F344 rats, Syrian golden hamsters and pigs and cloned flavin-containing monooxygenases (FMOs) from human and rabbit liver. Thermal inactivation at 45 degrees C for 2 min reduced FMO S-oxygenating activity but did not affect N-oxidation of NNK, leading to the conclusion that FMOs are not implicated in the detoxification of NNK. Detoxification of NNK was not increased by n-octylamine or by incubation at pH 8.4, supporting the conclusion that FMOs are not involved in the metabolism of NNK. SKF-525A (1 mM) significantly reduced N-oxidation and alpha-carbon hydroxylation, suggesting that these two pathways were catalyzed by cytochromes P450. Metabolism of NNK was lower with lung microsomes than with liver microsomes. Inhibition of metabolism of NNK by SKF-525A was also observed with rat lung microsomes, leading to the conclusion that cytochromes P450 are involved in pulmonary metabolism of NNK. Cloned FMOs did not metabolize NNK. In conclusion, cytochromes P450 rather than FMOs are involved in N-oxidation of NNK. The high capacity of hamster liver microsomes to activate NNK does not correlate with the resistance of this tissue to NNK-induced hepatocarcinogenesis.