Mono-allelic expression at the mouse IGF2/H19 locus is controlled by differential allelic DNA methylation of the imprinting control region (ICR). Because a randomly integrated H19 ICR fragment, when incorporated into the genome of transgenic mice (TgM), was allele-specifically methylated in somatic, but not in germ cells, it was suggested that allele-discriminating epigenetic signature, set within or somewhere outside of the Tg H19 ICR fragment in germ cells, was later translated into a differential DNA methylation pattern. To test if the chicken β-globin HS4 (cHS4) chromatin insulator might interfere with methylation imprinting establishment at the H19 ICR, we inserted the H19 ICR fragment, flanked by a set of floxed cHS4 core sequences, into a human β-globin locus YAC and generated TgM (insulated ICR' TgM). As controls, the cHS4 sequences were removed from one side (5'HS4-deleted ICR') or both sides (pseudo-WT ICR') of the insulated ICR' by in vivo cre-loxP recombination. The data show that while maternally inherited transgenic H19 ICR was not methylated in insulated ICR' TgM, it was significantly methylated upon paternal transmission, though the level was lower than in the pseudo-WT ICR' control. Because this reduced level of methylation was also observed in the 5'HS4-deleted ICR' TgM, we speculate that the phenotype is due to VEZF1-dependent demethylation activity, rather than the insulator function, borne in cHS4. Collectively, although we cannot rule out the possibility that cHS4 is incapable of blocking an allele-discriminating signal from outside of the transgene, the epigenetic signature appears to be marked intrinsically within the H19 ICR.