The Ly-6 Ag family consists of glycosyl-phosphatidylinositol-anchored surface proteins with a molecular mass of about 15 kDa. Seven members of the murine family have been characterized, and from five of these the genes have been cloned. Three members of the human family have been characterized: CD59, Ag E48, and the RIG-E or TSA-1/Sca-2 Ag. Most of the genes are expressed on lymphocytes, but some are expressed on other tissues as well. The mapped genes of the murine Ly-6 Ags, as well as of CD59, were shown to have a highly conserved structure, each consisting of four exons. The human E48 Ag was originally identified as a target Ag for radioimmunotherapy of patients with squamous cell carcinoma. The Ag is expressed on keratinocytes, but evidently not on lymphocytes. Molecular cloning of the cDNA encoding the Ag revealed that this Ag is most likely the human homologue of the murine Ly-6 Ag, ThB. In this paper, we describe that, in contrast to all other Ly-6 genes, the gene encoding the human E48 Ag consists of only three exons. Sequences at the 5' end of the transcription start site were shown to drive keratinocyte-associated expression. These data suggest that the functional elimination of an ancestral Ly-6 exon 1 switched the expression from lymphocytes toward keratinocytes.