We have previously shown that the distal promoter (promoter B) of the estrogen receptor alpha (ER alpha) gene is responsible for the enhanced expression of the ER alpha gene seen in human breast cancer and that a novel trans-acting factor, estrogen receptor promoter B associated factor 1 (ERBF-1), is required for transcription from promoter B in breast cancer cells. In development of breast cancer, loss of ER alpha gene expression is one of the most important steps in acquiring hormone resistance, though the mechanisms are poorly understood. Recent studies have reported that methylation of the ER alpha gene promoter A and exon 1 was inversely associated with ER alpha gene expression in human breast cancer and cell lines. The methylation status of the promoter B region, which is responsible for overexpression of ER alpha protein in cancer tissue, has not been investigated. In this report, we found that the methylation status of promoter B, as well as that of promoter A, was inversely associated with ER alpha gene expression in human breast cancer and cell lines. Specific methylation of ER alpha gene promoters in vitro directly decreased transcription of the ER alpha gene in a reporter assay. Demethylating treatment induced transcription of ER alpha mRNA from promoter B in ZR-75-1 cells, which showed no transcription from promoter B, despite weak ERBF-1 expression, but not in ER alpha-negative MDA-MB-231 and BT-20 cells, which lack ERBF-1. ZR-75-1 cells showed promoter activity equal to that of MCF-7 cells in a reporter assay. Our results indicate that methylation of promoter B of the ER alpha gene is important for loss of ER alpha gene expression in human breast cancer, and methylation of the promoters can directly modulate ER alpha gene expression. However, loss of critical transcriptional factors such as ERBF-1 may also be involved in some ER alpha-negative cases.