imprinted expression of the mouse igf2 h19 locus is controlled by parent of origin specific methylation of the imprinting control region icr we previously demonstrated that when placed in a heterologous genomic context the h19 icr fragment contains an intrinsic activity that allows it to acquire differential methylation in somatic cells but not in germ cells in the present study we investigated the requirements for the ctcf binding sites of the icr in the acquisition of post fertilization methylation to this end two mutant icr fragments were introduced into the human beta globin locus in a yeast artificial chromosome transgenic mouse tgm model 4xmut had mutations in all four icr ctcf binding sites that prevented ctcf binding but retained the methylation target cpg motifs and 9cg harbored mutations in the cpg motifs within the ctcf binding sites but each site retained constitutive ctcf binding activity in tgm germ cells and pre implantation blastocysts the absence of ctcf binding sites 4xmut did not lead to hypermethylation of the transgenic h19 icr however after implantation the mutations of ctcf sites 4xmut and 9cg affected the maintenance of methylation these results demonstrated that although the ctcf binding sites are indispensable for maintenance of the unmethylated state of the maternal icr in post implantation embryos they are not required to establish paternal allele specific methylation of the transgenic h19 icr in pre implantation embryos.