A tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is believed to contribute to the cancer burden in cigarette smokers. To evaluate NNK effects on the expression of lysyl oxidase (LOX), a tumor suppressor, we examined this enzyme at various levels in NNK-treated rat fetal lung fibroblasts (RFL6). Exposure of cells to NNK reduced levels of steady-states LOX mRNA and new transcript synthesis. NNK inhibited all LOX protein species in a dose-dependent manner. Although 300 µM NNK markedly decreased the level in the 46 kDa preproenzyme, under same conditions, there was no detectable amounts of the 50 kDa proenzyme and the 32 kDa mature enzyme suggesting NNK perturbing the LOX protein processing to its mature form. Moreover, NNK also suppressed LOX activities in conditioned media of treated cells. At the promoter level, NNK enhanced methylation of CpG, but decreased acetylation of histone H3 at the core promoter region of the LOX gene. These results indicated that transcriptional and translational processes of LOX are major targets for NNK. Thus, inactivation of tumor suppressor gene LOX may play a critical role in NNK carcinogenesis.