The imprinted mouse gene Gnas produces the G protein α-subunit G_Sα and several other gene products by using alternative promoters and first exons. G_Sα is maternally expressed in some tissues and biallelically expressed in most other tissues, while the gene products NESP55 and XLαs are maternally and paternally expressed, respectively. We investigated the mechanisms of Gnas imprinting. The G_Sα promoter and first exon are not methylated on either allele. A further upstream region (approximately from positions −3400 to −939 relative to the G_Sα translational start site) is methylated only on the maternal allele in all adult somatic tissues and in early postimplantation development. Within this region lies a fourth promoter and first exon (exon 1A) that generates paternal-specific mRNAs of unknown function. Exon 1A and G_Sα mRNAs have similar expression patterns, making competition between their promoters unlikely. Differential methylation in this region is established during gametogenesis, being present in oocytes and absent in spermatozoa, and is maintained in preimplantation E3.5d blastocysts. Therefore, this region is a methylation imprint mark. In contrast, differential methylation of the NESP55 and XLαs promoter regions (Nespand Gnasxl) is not established during gametogenesis. The methylation imprint mark that we identified may be important for the tissue-specific imprinting of G_Sα.