Colon cancer incidence and mortality rates are lower in females compared with males, and numerous epidemiological studies suggest that estrogen replacement therapy (ERT) reduces cancer risk in postmenopausal women. Two estrogen receptor (ER) subtypes, ERα and ERβ, mediate genomic effects in target cells. The aim of this study was to determine the relative mRNA expression levels for ER subtypes and ERβ isoforms in colon tumors, normal colonic mucosa, and colon cancer cell lines. ERαand ERβ isoform mRNA levels were investigated in paired samples of colon tumors and normal mucosa from 26 patients using comparative reverse transcription-PCR and then Southern analyses. Constitutive steroid hormone receptor mRNA levels were determined for five colon adenocarcinoma cell lines using reverse transcription-PCR,and ERβ levels were further studied in Caco-2 cells using Northern and Western analyses. ERβ mRNA steady-state levels (relative to glyceraldehyde-3-phosphate dehydrogenase mRNA) were significantly decreased in colon tumors compared with normal mucosa in female patients. ERβ1 and ERβ2 isoform mRNA levels were significantly decreased in tumors from female patients, and ERβ1 mRNA levels were also significantly lower in tumors from female patients compared with tumors from males. ERα mRNA levels were much lower than ERβ levels and were similar between normal mucosa and tumor samples in both genders. ERβ mRNA was detected in Caco-2, T84, and SW1116 cell lines and all lines were essentially negative for ERα mRNA. Caco-2 cells coexpressed ERβ1, ERβ2, and ERβ5 mRNA, though a single protein transcript was observed. ERβ protein was detected in normal colonic superficial epithelium, vascular smooth muscle and endothelium, and enteric neurons by immunohistochemistry. These data show that ERβ is the predominant ER subtype in the human colon and that decreased levels of ERβ1 and ERβ2 mRNA are associated with colonic tumorigenesis in females. This information suggests that activation of ERβ-mediated processes in the superficial colonic epithelium may have a role in the preventive effects observed for female gender and ERT usage.