In mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ( and references therein). The currently most extensively studied examples of this phenomenon, termed genomic imprinting, are the physically linked Igf2 (insulin-like growth factor II) and H19 genes, which are expressed mono-allelically from opposite parental alleles  . The repressed status of the maternal Igf2 allele is due to cis elements that prevent the H19 enhancers  from accessing the Igf2 promoters on the maternal chromosome  . A differentially methylated domain (DMD) in the 5' flank of H19 is maintained paternally methylated and maternally unmethylated  . We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF (  and references therein) to the H19 DMD is methylation-sensitive and parent of origin-dependent. Selectively mutating CTCF-contacting nucleotides, which were identified by methylation interference within the extended binding sites initially revealed by nuclease footprinting, abrogated the H19 DMD enhancer-blocking property. These observations suggest that molecular mechanisms of genomic imprinting may use an unusual ability of CTCF to interact with a diverse spectrum of variant target sites, some of which include CpGs that are responsible for methylation-sensitive CTCF binding in vitro and in vivo.