Estrogen receptor-α (ER-α) plays a crucial role in normal breast development and has also been linked to mammary carcinogenesis and clinical outcome in breast cancer patients. However, ER-α gene expression can change during the course of disease and, consequently, therapy resistance can occur. The molecular mechanism governing ER-α transcriptional activity and/or silencing is still unclear. Here, we showed that the presence of a specific pRb2/p130 multimolecular complex on the ER-α promoter strongly correlates with the methylation status of this gene. Furthermore, we suggested that pRb2/p130 could cooperate with ICBP90 (inverted CCAAT box binding protein of 90 kDa) and DNA methyltransferases in maintaining a specific methylation pattern of ER-α gene. The sequence of epigenetic events for establishing and maintaining the silenced state of ER-α gene can be locus- or pathway- specific, and the local remodeling of ER-α chromatin structure by pRb2/p130 multimolecular complexes may influence its susceptibility to specific DNA methylation. Our novel hypothesis could provide a basis for understanding how the complex pattern of ER-α methylation and transcriptional silencing is generated and for understanding the relationship between this pattern and its function during the neoplastic process. [Cancer Res 2007;67(16):7731–7]