The effects of both chronic ethanol consumption and repeated exercise on metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by rat lung microsomes were investigated. Keto aldehyde production was significantly enhanced 52% and 98%, respectively, by the ethanol and exercise, as was keto alcohol production (72% and 76%). 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) production was significantly enhanced only by the exercise (+32%). The combined treatment enhanced the keto alcohol production by 39%, indicating non-additive effects on cytochrome P450 (CYP) isozymes by the different treatments. Immunoblot and metabolic studies revealed an increased activity and content of CYP1A2 and CYP2B induced by the ethanol and exercise treatments as well as an induction of CYP2E1 proteins by the ethanol and combined treatments. These results indicate that both ethanol consumption and running exercise enhance NNK activation by increasing the expression of the CYP enzymes responsible for NNK activation.