Aberrant DNA methylation is a major epigenetic mechanism of gene silencing in a wide range of human cancers. Previous studies on DNA methylation typically used paired tumor and normal-appearing surrounding tissues from cancer-bearing individuals. However, genomic DNA isolated from surrogate tissues such as blood cells represents an attractive material that can be exploited in the discovery of biomarkers of exposure and tumorigenesis. Here we examined the association between lung cancer and DNA methylation patterns in a panel of candidate genes. We also investigated whether blood levels of vitamin metabolites modify DNA methylation levels in blood cells. To this end, we quantitatively determined DNA methylation levels in blood cells of nested cases and controls from a prospective study with well defined dietary habits and lifestyles. Multiple CpG sites in five genes (CDKN2A/p16, RASSF1A, GSTP1, MTHFR, and MGMT) that are frequent targets of hypermethylation in a variety of human malignancies were included in the analysis. While no clear association between DNA methylation patterns and the case/control status was found, with the exception of RASSF1A hypermethylation, methylation level changed according to serum levels of 1-carbon metabolites and vitamins B. Overall, folate was associated with increased methylation levels of RASSF1A and MTHFR and methionine was associated with decreased methylation levels of RASSF1A. The associations with folate were more pronounced among never smokers while the associations with methionine were more evident among ever-smokers. These results are consistent with the notion that blood levels of 1-carbon metabolism markers and dietary/lifestyle factors may modify DNA methylation levels in blood cells and that blood cells can be exploited for the discovery of epigenetic biomarkers of exposures, providing proof-of-principle on the use of blood samples in the context of prospective studies.