In the mouse Igf2/H19 imprinted locus, differential methylation of the 
imprinting control region (H19 ICR) is established during spermatogenesis and is 
maintained in offspring throughout development. Previously, however, we observed 
that the paternal H19 ICR, when analyzed in yeast artificial chromosome 
transgenic mice (YAC-TgM), was preferentially methylated only after 
fertilization. To identify the DNA sequences that confer methylation imprinting, 
we divided the H19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both 
ends of a λ DNA fragment into which CTCF binding sites had been inserted, and 
analyzed this in YAC-TgM. The maternally inherited λ sequence, normally 
methylated after implantation in the absence of H19 ICR sequences, became 
hypomethylated, demonstrating protective activity against methylation within the 
ICR. Meanwhile, the paternally inherited λ sequence was hypermethylated before 
implantation only when a 1.7-kb fragment was ligated. Consistently, when two 
subfragments of the H19 ICR were individually investigated for their activities 
in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal 
allele-specific DNA methylation. These results show that postfertilization 
methylation imprinting is conferred by a paternal allele-specific methylation 
activity present in a 1.7-kb DNA fragment of the H19 ICR, while maternal 
allele-specific activities protect the allele from de novo DNA methylation.