BACKGROUND Folate deficiency decreases thymidylate synthesis from deoxyuridylate, which results in an imbalance of deoxyribonucleotide that may lead to excessive uracil misincorporation (UrMis) into DNA during replication and repair. OBJECTIVE We evaluated the relation between UrMis in different tissues and the effect of folate supplementation on UrMis. METHODS We analyzed UrMis concentrations in rectal mucosa (n = 92) and white blood cells (WBCs; n = 60) among individuals randomly assigned to receive supplementation with 1 mg folate/d or placebo, who were then evaluated for colorectal adenoma recurrence. RESULTS As expected, total homocysteine was significantly lower among the study participants who received active folate treatment (Wilcoxon's P = 0.003) than among those in the placebo group. The median UrMis concentration in rectal mucosa and WBCs among individuals treated with folate was not significantly lower than that in those who received placebo (Wilcoxon's P = 0.17). UrMis concentrations in both rectal mucosa and WBCs did not correlate significantly with folate measured in plasma and red blood cells. UrMis in rectal mucosa was marginally associated with an increased risk of adenoma recurrence (odds ratio per SD: 1.43; 95% CI: 0.91, 2.25). CONCLUSIONS UrMis measurements in WBCs are not a robust surrogate for UrMis measurements in the rectal mucosa (Spearman correlation coefficient = 0.23, P = 0.08). Furthermore, folate supplementation in an already replete population (half treated with folic acid supplements and all exposed to folic acid fortification of the food supply) was not significantly associated with reduced UrMis in rectal mucosa cells or WBCs. Large-scale studies are needed to evaluate whether excessive UrMis concentrations are an important risk factor for colorectal neoplasia. This trial was registered at clinicaltrials.gov as NCT00272324.