Two novel estrogen receptor beta (ERbeta) mRNA isoforms that diverge in their 5'-untranslated regions, ERbeta mRNA (0K-1) and ERbeta mRNA (0N-1), have recently been identified. This indicates that transcription of the human ERbeta gene occurs from at least two different promoters, named promoter 0K and promoter 0N. The aim of this study was to investigate the expression of ERbeta isoforms in primary cultures of normal breast epithelial cells, a panel of breast cancer cell lines and in normal breast and breast cancer tissues; and to examine whether methylation of the two ERbeta promoters is involved in regulation of ERbeta gene expression. Using quantitative real-time PCR techniques, we found that ERbeta mRNA levels were significantly lower in breast cancer cell lines than in primary cultures of normal breast epithelial cells. Bisulfite genomic sequencing analysis revealed that two promoters of the ERbeta gene exhibit distinct methylation patterns. Promoter 0N was unmethylated in normal breast epithelial cells, but extensively methylated in breast cancer cell lines. In contrast, promoter 0K was unmethylated in both normal and malignant breast epithelial cells. We demonstrated a significant correlation between promoter 0N hypermethylation and loss of ERbeta mRNA expression in breast cancer cell lines. Treatment of breast cancer cells with demethylating agent effectively reactivated the expression of ERbeta mRNA. The observations from the cell lines were also reflected in primary breast cancer tumors. Thus, expression of ERbeta mRNA in breast tumors was found to be inversely associated with the degree of methylation of promoter 0N. Our results suggest that a decreased level of ERbeta mRNA may be associated with breast tumorigenesis, and that DNA methylation is an important mechanism for ERbeta gene silencing in breast cancer.