CD53 is a pan-leukocyte surface glycoprotein which spans the plasma membrane four times and is a member of the transmembrane 4 superfamily (TM4SF). The protein sequence and gene structure of mouse CD53 (Cd53) were determined by isolation of both genomic and cDNA clones. CD53 is highly conserved in evolution, as mouse Cd53 was 91% identical to rat CD53 and 82% identical to human CD53. The mouse Cd53 gene spanned approximately 9.0 kb of DNA and encoded the 219 amino acid residues of CD53 over seven exons. The Cd53 gene produced a 1.8 kb transcript which was dramatically upregulated after cell activation. The mouse Cd53 gene was mapped to chromosome 3, whereas the locus of another TM4SF member, CD37 (Cd37), was mapped to mouse chromosome 7. Three lines of evidence suggest that the TM4SF arose divergently from an ancestral gene. First, the gene structure of CD53 was strikingly similar to two other members of the TM4SF, CD63 and TAPA-1; second, Cd37 mapped to the same chromosome as Tapa-1; and, third, Cd37 mapped to a segment of chromosome 7 that contains a number of genes that are structurally or functionally related to genes closely linked to Cd53 on chromosome 3.