Abstract. DNA methylation is a heritable and reversible modification to CpG sites in the mammalian genome. Parental allele-specific methylation is hypothesized to be important in the establishment and maintenance of imprinted gene expression; however, dynamic changes in allele-specific patterns have been observed. The upstream regulatory region of the small nuclear riboprotein N gene (SNRPN) is an important imprinting control region (ICR) for establishing and maintaining the methylation imprint in the locus on 15q1113 associated with Prader-Willi and Angelman syndromes (PWS). To compare directly the role of allele-specific methylation patterns and the maintenance of imprinted expression in the PWS region, clonal populations of normal T lymphocytes were cultured for 2225 generations. A novel long-range semi-nested polymerase chain reaction (PCR) strategy was utilized in order to span two different methylation sites, and a polymorphism within SNRPN was used so that allele-specific methylation of both sites could be determined. Reverse transcription/PCR followed by polymorphism analysis was also performed in order to determine parental allele-specific transcription. Exclusive paternal expression at both SNRPN and IPW was maintained in all T cell clones and correlated with maternal methylation of the intron1 NotI site. In contrast, biallelic methylation was observed in all clones at the previously described paternally methylated HpaII site in intron7. These results demonstrate that the maintenance of paternal expression of SNRPN and IPW correlates with a strict clonal maintenance of allele-specific methylation at the CpG-dense 5' end of SNRPN. Differential maintenance of methylation sites within imprinted genes may depend on the density and chromatin organization of surrounding CpG sites.