Colorectal cancer (CRC) is the second most common cancer, in both sexes, in developed countries and the incidence rates are rising in many populations. Less than 10% of cases are thought to be due to recognised familial syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC). Epidemiological studies suggest that dietary factors play an important role in the aetiology of CRC; disease incidence is inversely associated with diets high in folate and has been positively associated with red meat consumption. Folate is important in DNA methylation and synthesis. The increased risk associated with red meat is proposed to be due to the formation of heterocyclic amines during cooking rather than meat consumption per se. The current study, a population-based case-control study (269 case and 408 control subjects) was carried out in the Grampian region and investigated polymorphisms in genes involved in folate and xenobiotic metabolism. Inter-individual differences in the activation and detoxification of xenobiotics and the metabolism of folate might alter risk of CRC. Mouthwash samples were collected from all participants and genomic DNA was extracted for genotypic analysis of MTHFR, CYP1A1, NAT2, GSTM1 and GSTT1. Homozygous possession of the MTHFR A1298C substitution was associated with a reduced risk of colon cancer. The reduced risk of rectal cancer associated with possession of the MTHFR C677T substitution approached statistical significance. A proposed mechanism is presented to explain the inverse association between CRC and MTHFR allelic variants. The CYP1A1 C2453A substitution was inversely associated with CRC risk. No significant alteration of risk was associated with deletions of GSTM1 or GSTT1 genes or acetylation status imputed from NAT2 genotype. Genomic DNA extracted from mouthwash samples, collected by post from an elderly population, was effective as template for PCR/RFLP methods of genotyping.