Loss of heterozygosity (LOH) and/or inactivation of tumor suppressor genes are implicated in the initiation and progression of many malignancies, including colorectal cancer. Although accumulating evidence suggests a chemopreventive role for folate in colorectal cancer, regulatory mechanisms are poorly understood. The primary objective of the current investigation was to determine whether folic acid would prevent LOH of the three tumor suppressor genes, deleted in colorectal cancer (DCC), adenomatous polyposis coli (APC) and p53 in macroscopically normal appearing rectal mucosa of patients with adenomatous polyps. In addition, the effect of folic acid on rectal mucosal proliferation was determined. Twenty patients were randomized in a double-blind study to receive either folic acid 5mg once daily or identical placebo tablets for 1 year. Genomic DNA and total protein were extracted from the rectal mucosa at baseline and after 1 year of treatment and analyzed for LOH and protein levels of APC, DCC and p53 genes. In addition, paraffin-embedded mucosal specimens were analyzed for proliferating cell nuclear antigen (PCNA) immunoreactivity, as a measure of cellular proliferative activity. Folate supplementation prevented LOH of DCC gene in five out of five (100%) patients who demonstrated baseline heterozygosity, whereas two out of four (50%) placebo-treated patients with baseline heterozygosity demonstrated allelic loss. Mucosal protein levels of DCC were also reduced in 7 of 10 (70%) placebo-treated patients compared to only 2 of 10 (20%) of patients treated with folate. Levels increased, however, in eight and three patients in the folic acid and placebo groups, respectively (P<0.02). Folic acid caused no change in allelic status of either APC or p53 gene. Folate supplementation caused a small, but not statistically significant, 16% reduction in mucosal proliferation, whereas placebo treatment resulted in a 88% (P<0.05) increase in this parameter, when compared with the corresponding baseline values. Our results indicate that folic acid prevents an increase in proliferation and arrests LOH of DCC gene and also stabilizes its protein in normal appearing rectal mucosa of patients with colorectal adenomas. Taken together, our data suggest that one of the ways folate may exert its chemopreventive effect is by stabilizing certain tumor suppressor gene(s) and preventing further increases in proliferation.