Epidemiological studies have demonstrated a causal link between tobacco smoking and lung cancer. We investigated the association between inactivation of the p16 gene and tobacco smoking in 51 non-small cell lung cancers (NSCLCs). Aberrations of the p16 gene were studied by PCR single-strand conformation polymorphism analysis, followed by direct sequencing, microsatellite analysis, methylation-specific PCR, and immunohistochemistry. Mutations were detected in 3.9% (2/51) of the tumors; the tumors carrying mutations were from smokers. The incidences of loss of heterozygosity, homozygous deletion, and promoter methylation in 37 smokers vs. 14 non-smokers were; 45.9% vs. 28.6%, 16.2% vs. 7.1%, and 35.1% vs. 7.1%, respectively. Among these, only the association between promoter methylation and tobacco smoking was statistically significant (P < 0.05). Therefore, epigenetic aberration is considered to be a major causative event in p16 silencing by tobacco smoking. Loss of p16 protein expression was apparent in 49% (25/51) of the tumors, and was associated with tobacco smoking (P < 0.05) and with histological type (P < 0.05). These findings suggest that tobacco smoking leads to inactivation of the p16 gene mainly through the epigenetic mechanism, ultimately increasing the risk of NSCLC, especially the squamous cell histological type.