The Ly9 glycoprotein is a member of the immunoglobulin (Ig) superfamily, which is expressed on the cell surface of B and T lymphocytes. With two alleles (Ly9.1 and Ly9.2), it was first described as an alloantigenic marker of lymphocyte differentiation. Ly9 consists of four Ig-like domains with the structural features of the CD2 subfamily, which includes CD2, CD48, CD58, 2B4, CD84, and CDw15O (SLAM). Here, we report the isolation and characterization of the Ly9 gene, which encompasses at least 19 kb and contains ten separated exons, with sizes ranging from 54 to 355 bp. Each Ig-like domain is encoded by an individual exon. Sequence analysis of a 1.5-kb fragment upstream from the start translational codon revealed the absence of appropriately located TATA and CAAT boxes. However, potential binding sites for the transcription factors PU.1, Ikaros, AP-1, GATA-2, NF-GMa, NFAT-1, and Oct-2, which are involved in the early development and maturation of lymphocytes, were found. To further characterize the two allotypes of Ly9, cDNA of Balb/C and C57BL/6 mouse strains were sequenced and the predicted polypeptides compared. Nine discrepancies were found, four of them in the first Ig-like domain. The characterization of the genomic organization of Ly9 presented in this paper may improve understanding of the molecular mechanisms that regulate Ly9 expression, and the production of a construct to disrupt the Ly9 gene in ES cells in order to produce deficient mice.