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The down-regulation of the estrogen receptor-β (ERβ) gene is associated with several malignancies, including prostate carcinoma. The purpose of the current study was to investigate the mechanisms of ERβ inactivation through the analysis of CpG methylation of the promoter region of ERβ gene. ERβ protein expression was examined by immunohistochemistry in 23 cases of human prostate carcinoma and 40 cases of benign prostatic hyperplasia (BPH). DNA was extracted from these tissues and processed for sodium bisulfite genomic sequencing. The percentage of methylation of CpG sites in the promoter region of ERβ (−376 to −117), which contains 19 CpG sites, was determined from genomic sequencing data. The prostate carcinoma cell lines DU145 and ND1 were treated with the demethylating agent 5-AZAC and ERβ mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction. In BPH tissues, ERβ protein expression was found mainly in epithelial cells. ERβ protein expression was lacking in 83% of prostate carcinoma samples (19 of 23 samples) whereas all cases of BPH (40 of 40) demonstrated expression of ERβ protein. The mechanism of inactivation of the ERβ gene in prostate carcinoma was CpG methylation because the degree of methylation at all CpG sites within the promoter region between −376 and −117 was higher in prostate carcinoma samples compared with BPH tissues. Nine of 19 CpG sites within the promoter region of ERβ displayed significant differences in methylation between prostate carcinoma and BPH samples. The prostate carcinoma cell lines appeared to lack ERβ expression. However, 5-AZAC treatment restored ERβ expression in those cell lines, suggesting that methylation inactivates the ERβ gene in prostate carcinoma. The results of the current study demonstrate, for what we believe to be the first time, that the inactivation of the ERβ gene in prostate carcinoma occurs through CpG methylation of the promoter region of this gene. Cancer 2001;92:2076–83. © 2001 American Cancer Society.