Abstract CpG island methylation in the promoter regions of tumor suppressor genes has been shown to occur in normal colonic tissue and can distinguish between subjects with and without colorectal neoplasms. It is unclear whether this relationship exists in other tissues such as blood. We report the relationship between estrogen receptor gene (estrogen receptor α) methylation in leukocyte and normal colonic tissue DNA in subjects with and without colorectal neoplasia. DNA was extracted from frozen stored whole blood samples of 27 subjects with cancer, 30 with adenoma, 16 with hyperplastic polyps, and 57 disease-free subjects. DNA methylation in seven CpG sites close to the transcription start of estrogen receptor α was quantitated using pyrosequencing and expressed as a methylation index (average methylation across all CpG sites analyzed). Estrogen receptor α methylation in leukocyte DNA was compared with estrogen receptor α methylation in normal colonic mucosa DNA that had been previously determined in the same subjects. Estrogen receptor α was partially methylated (median, 4.3%; range, 0.0-12.6%) in leukocyte DNA in all subjects, with no significant difference between disease groups (P > 0.05). Estrogen receptor α methylation in leukocytes was 60% lower than estrogen receptor α methylation in normal colonic tissue (P < 0.001). Estrogen receptor α methylation in colonic tissue (P < 0.001) and smoking (P = 0.016) were determinants of estrogen receptor α methylation in leukocytes, independent of age, body mass index, gender, and disease status. In conclusion, there was a positive relationship between estrogen receptor α methylation in leukocytes and colonic tissue in subjects with and without colorectal tumors. However, unlike in colonic tissue, estrogen receptor α methylation in leukocytes was unable to distinguish between disease groups. (Cancer Epidemiol Biomarkers Prev 2009;18(3):922–8)