MacroH2A1 is a histone variant that has been previously known to be enriched in the inactive X chromosome of female mammals. This histone variant is closely associated with the repressed regions of chromosomes. In this study, the deposition patterns of macroH2A1 at a number of different genomic loci located in X chromosome and autosomes were analyzed. MacroH2A1 is preferentially deposited at methylated CpG-rich regions located close to promoters. The macroH2A1 deposition patterns at the methylated CpG islands of several imprinted domains, including the Imprinting Control Regions (ICRs) of Xist, Peg3, H19/Igf2, Gtl2/Dlk1, and Gnas domains, show consistent allele-specificity towards inactive, methylated alleles. The macroH2A1 deposition levels at the ICRs and other Differentially Methylated Regions (DMRs) of these domains are also either higher or comparable to those observed at the inactive X chromosome of female mammals. The results indicate that, besides DNA methylation, macroH2A1 is another epigenetic component in the chromatin of ICRs displaying differential association with two parental alleles. The potential roles of macroH2A1 in genomic imprinting were investigated by lowering the cellular levels of the macroH2A1 protein using siRNA technique. The expression levels of a subset of genes, including Peg3 and Usp29 of the Peg3 domain, and Nesp of the Gnas domain were changed in macroH2A1 knockdown Neuro2A cells. The expressions of these genes were further down-regulated, but not up-regulated, in response to reduced protein levels of the potential repressor macroH2A1. This down-regulation was not accompanied with changes in the DNA methylation status of these domains. This suggests that macroH2A1 may not function as a dominant transcriptional repressor for most imprinted genes, but that macroH2A1 may participate in the heterochromatin formation of imprinted domains with functions yet to be discovered.