CD1 differentiation antigens are defined by a group of monoclonal antibodies that characterize immature human thymocytes. A cloned cDNA has been used to identify CD1 genes in a human genomic library. Five CD1 genes have been isolated, and Southern blot analysis suggests that these represent all the cross-hybridizing human CD1 genes. They share a highly conserved exon, which is homologous to the beta 2-microglobulin-binding domain (alpha 3) of major histocompatibility complex (MHC) class I antigens. In this domain, amino acid sequences are 71-88% homologous. However, the homology between CD1 and MHC class I alpha 3 domains is only 21%. This is the same degree of homology as between either of them and the class II beta 2 domain, which does not bind beta 2-microglobulin. The evolutionary implications of these results are discussed.