Recent studies from our laboratory suggest that gene-specific methylation changes in sputum could be good intermediate markers for the early detection of lung cancer and defining the efficacy of chemopreventive interventions. The purpose of our study was to determine the prevalence for aberrant promoter methylation of the p16, O(6)-methylguanine-DNA methyltransferase (MGMT), death-associated protein (DAP) kinase, and Ras effector homologue (RASSFIA) genes in nonmalignant bronchial epithelial cells from current and former smokers in a hospital-based, case control study of lung cancer. The relationship between loss of heterozygosity, at 9p and p16 methylation in bronchial epithelium and the prevalence for methylation of these four genes in sputum from cancer-free, current and former smokers were also determined. Aberrant promoter methylation of p16 was seen in at least one bronchial epithelial site from 44% of cases and controls. Methylation of the DAP kinase gene was seen in only 1 site from 5 cases and 4 controls, whereas methylation of the RASSFIA was not detected in the bronchial epithelium. Promoter methylation for p16 and DAP kinase was seen as frequently in bronchial epithelium from current smokers as from former smokers. No promoter methylation of these genes was detected in bronchial epithelium from never-smokers. Methylation of the p16 gene was detected in sputum from 23 of 66 controls. DAP kinase gene promoter methylation was also seen in sputum from 16 controls, and 8 of these subjects were positive for p16 methylation. Methylation of the MGMT gene was seen in sputum from 9 controls, whereas RASSFIA promoter methylation was only seen in 2 controls. The correlation between p16 status in the bronchial epithelium obtained from lung lobes that did not contain the primary tumor and the tumor itself was examined. Seventeen of 18 tumors (94%) showed an absolute concordance, being either methylated in the tumor and at least 1 bronchial epithelial site, or unmethylated in both tumor and bronchial epithelium. These results indicate that aberrant promoter hypermethylation of the p16 gene, and to a lesser extent, DAP kinase, occurs frequently in the bronchial epithelium of lung cancer cases and cancer-free controls and persists after smoking cessation. The strong association seen between p16 methylation in the bronchial epithelium and corresponding primary tumor substantiates that inactivation of this gene, although not transforming by itself, is likely permissive for the acquisition of additional genetic and epigenetic changes leading to lung cancer.