We have evaluated the effect of folate supplementation (5 mg/day) on global deoxyribonucleic acid (DNA) methylation status of the rectal mucosa of 20 patients with resected colonic adenomas in a prospective, controlled, cross-over study. Baseline values of DNA methylation were inversely correlated with caloric (P = 0.03) and fat intake (P = 0.05) and patients harbouring multiple polyps consumed significantly more calories (P = 0.0006), fat (P = 0.009) and carbohydrates (P = 0.009) as compared to patients having one single lesion. Folate supplementation resulted in a significant decrease of DNA hypomethylation in 7/20 patients (P = 0.05) which returned to previous values after placebo treatment. This effect was significantly correlated with number of polyps, with all the responders presenting one single lesion, whereas 8/13 of the non-responders had multiple ones (chi2 = 7.17, P = 0.007). In conclusion, folate supplementation may decrease degree of DNA hypomethylation, but only in patients with one single polyp. In those with multiple lesions, other nutritional factors such as caloric and fat intake, may be more determinant.