Previous annotation of the Class III region of the human Major Histocompatibility Complex (MHC) depicts NG36 as an independent gene, which lies immediately centromeric to the G9a gene. However, data presented in this report show that in human and mouse cells the NG36 and G9a genes are predominantly expressed within a single approximately 3.9-kbp transcript. Thus, the human NG36/G9a gene contains 28 exons (4 exons from the NG36 gene and 24 exons from the G9a gene), spans 17.938 kb, and encodes a 1210-amino acid polypeptide. In addition, a splice variant (NG36G9a-SPI), which lacks exon 10, was found to be coexpressed together with the full-length NG36/G9a transcript in both human and mouse cells. To aid functional characterization of the novel NG36/G9a gene-product, T7-epitope-tagged versions of the complete NG36/G9a protein or the G9a region alone (amino acids 210 to 1210) was transiently expressed in mammalian cells. Surprisingly, the sub-cellular distribution of the NG36/G9a-T7 and G9a-T7 proteins was found to be quite distinct. Whereas the G9a-T7 protein was observed in both the cytoplasm and the nucleus, the NG36/G9a-T7 protein was extensively concentrated within the nucleus. Also, the G9a-T7 protein frequently appeared marginalized at the nuclear periphery, while the NG36/G9a-T7 protein was generally found throughout the nucleoplasm. As such, it would appear that the NG36 domain plays a key role in controlling the sub-cellular distribution of the NG36/G9a protein.