The silencing of tumor suppressor genes (TSGs) by aberrant hypermethylation occurs frequently in human cancer. Recently the RUNX3 gene was identified as a TSG inactivated by hypermethylation. We examined RUNX3 expression by reverse transcription-PCR and the methylation status of this gene by methylation specific-PCR in 43 lung cancer cell lines and 120 primary non-small cell lung cancer (NSCLC) tumor samples. RUNX3 expression was absent in 10 (50%) of 20 small cell lung cancer (SCLC) cell lines, 8 (50%) of 16 adenocarcinoma (AdC) cell lines, and 1 (33.3%) of 3 squamous cell carcinoma (SqC) cell lines. The frequency of RUNX3 methylation was significantly higher in AdC (7/16, 43.8%) than SCLC cell lines (1/20, 5%; p=0.032). RUNX3 expression was restored by treatment with 5-aza-2'-deoxycytidine and/or trichostatin-A in AdC cell lines. These results indicated that RUNX3 expression was regulated by aberrant hypermethylation in AdC cell lines. RUNX3 methylation was detected in 30 (25%) of 120 primary NSCLC tumors. RUNX3 methylation was significantly more frequent in non-smokers (16/43, 37.2%) than smokers (12/71, 16.9%; p=0.014), and in patients with AdC (26/72, 36.1%) than in patients with SqC (3/45, 6.7%; p<0.001). These results indicated that silencing of the RUNX3 gene plays an important role in the pathogenesis of lung cancer, and aberrant methylation is an important mechanism of inactivation of the RUNX3 gene in lung AdC.