Older age and inadequate folate intake are strongly implicated as important risk factors for colon cancer and each is associated with altered DNA methylation. This study was designed to determine the effects of aging and dietary folate on select features of DNA methylation in the colon that are relevant to carcinogenesis. Old (18 mo; n = 34) and young (4 mo; n = 32) male C57BL/6 mice were randomly divided into 3 groups and fed diets containing 0, 4.5, or 18 mumol folate/kg (deplete, replete, and supplemented groups, respectively) for 20 wk. Genomic DNA methylation and p16 promoter methylation in the colonic mucosa were analyzed by liquid chromatography/electrospray ionization/MS and methylation-specific PCR, respectively. p16 gene expression was determined by real-time RT-PCR. Old mice had significantly lower genomic DNA methylation compared with young mice at each level of dietary folate (4.5 +/- 0.2, 4.8 +/- 0.1, and 4.9 +/- 0.1 vs. 6.0 +/- 0.1, 5.3 +/- 0.2, and 5.9 +/- 0.2%, in folate-deplete, -replete, and -supplemented groups, respectively, P < 0.05) and markedly higher p16 promoter methylation (61.0 +/- 2.7, 69.7 +/- 6.9, and 87.1 +/- 13.4 vs. 10.8 +/- 3.6, 8.4 +/- 1.8, and 4.9 +/- 1.7%, respectively, P < 0.05). In old mice, genomic and p16 promoter DNA methylation each increased in a manner that was directly related to dietary folate (P(trend) = 0.009). Age-related enhancement of p16 expression occurred in folate-replete (P = 0.001) and folate-supplemented groups (P = 0.041), but not in the folate-deplete group. In conclusion, aging decreases genomic DNA methylation and increases promoter methylation and expression of p16 in mouse colons. This effect is dependent on the level of dietary folate.