We measured the concentrations of folate and vitamin B-12 in paired tissue samples of squamous cell cancer (SCC) and adjacent grossly normal-appearing uninvolved bronchial mucosa (from which SCC developed and also "at risk" of developing SCC) of the lung in 12 subjects to determine the involvement of these vitamins in 1) lung carcinogenesis and 2) global DNA methylation. The folate concentrations were significantly lower in SCCs than in uninvolved tissues (p = 0.03). The vitamin B-12 concentrations were also significantly lower in SCCs than in uninvolved tissues (p = 0.02). The radiolabeled methyl incorporation (inversely related to the degree of in vivo DNA methylation) was significantly higher in SCCs than in uninvolved tissues (p < 0.0001). The correlation between folate and radiolabeled methyl incorporation was inverse and statistically significant in SCCs (p = 0.03). The correlation between vitamin B-12 and radiolabeled methyl incorporation also was inverse and statistically significant in SCCs (p = 0.009). The relationship between tissue vitamin B-12 and DNA methylation was minimal in uninvolved tissues. The relationship between folate and DNA methylation, however, was inverse in uninvolved tissues. In the multiple regression models that included both vitamins, only folate was inversely associated with radiolabeled methyl incorporation in uninvolved and cancerous tissues. These results suggested that folate might be the limiting vitamin for proper DNA methylation in SCC as well as in tissues at risk of developing SCC. Several possible mechanisms of folate deficiency, including inactivation of the vitamin by exposure to carcinogens of cigarette smoke and underexpression or absence of folate receptor in SCCs and associated premalignant lesions, are discussed in light of these findings.