The chicken embryonic β-type globin gene, ρ, is silenced on day five of embryogenesis. Concomitant with this silencing is methylation of cytosine residues in the promoter and proximal transcribed region of the gene, which is first detected on day seven and is complete in adult cells. Once methylated, expression of the gene cannot be induced unless the methylation is removed by treatment of cells with Sazacytidine. Therefore ρ-globin is a member of a small group of genes whose normal developmentally regulated expression is mediated at least in part by DNA methylation.A methyl-DNA binding complex, termed the MeCPC (Erythroid Methyl Cytosine-binding Protein Complex), has been found to bind to the methylated, but not unmethylated, ρ-globin promoter and proximal transcribed region in nuclear extracts from definitive erythrocytes. This complex has a stronger binding affinity for its cognate binding sequence, the methylated ρ-globin proximal transcribed region (M-ρ248), than for an artificial 5-methylcytosine-rich sequence (M-CG11).To define the components of the MeCPC, we developed two chromatographic procedures to purify the complex from adult chicken red blood cell nuclear extracts (Purification Strategies I and II). Mass spectrometry was performed on the MeCPC obtained by Purification Strategy I and proteins were identified by a novel application of peptide mass fingerprint data fitting. Four components of the previously-purified MeCPl transcriptional repression complex were identified in the sample: MBD2, RbAp48, HDAC2 and MTA1. Another identified protein, MENT, is a factor expressed only in chicken hematopoietic cells. These five proteins, as well as the MeCPl component Mi2, were found to tightly coelute by Western blotting of gel-filtration fractions from Purification Strategy II. Therefore, we conclude that these five proteins are components of the MeCPC.To confirm that MBD2 is associated with the ρ-globin gene in vivo, we perfomed the chromatin immunoprecipitation assay using anti-MBD2 antibodies. In adult erythrocytes, significant enrichment for MBD2 is seen at the transcriptionally inactive ρ-globin gene, but no enrichment is observed at the transcriptionally active βA globin gene. These experiments confirm that MBD2 binds to the methylated p-globin gene in adult chicken erythroid cells.