A notable feature of Ly-5, among immunogenetic systems that identify glycoproteins of the cell surface and define the surface phenotype of cells according to their lineage, is that the Ly-5 locus specifies a range of molecular isoforms that distinguish cells of different stages and branches of hematopoietic development. The composition of the Ly-5 locus is of much interest in regard to how these isoforms are constructed and differentially regulated according to cell lineage. We describe here a cDNA clone, pLy-5-68, that identifies Ly-5. The Ly-5 specificity of the pLy-5-68 clone was first indicated by a restriction fragment length polymorphism (RFLP), which in Southern blotting distinguishes genomic DNA of C57BL/6 (B6) mice (Ly-5a) from that of B6-Ly-5b congeneic mice whose genome is the same as B6 except for the segment of chromosome 1 that bears Ly-5b. For the following reasons it is unlikely that pLy-5-68 represents a gene linked to Ly-5 that was carried over with Ly-5b during serial backcrossing to make the B6-Ly-5b congeneic strain. In all mouse strains tested, the serological Ly-5 allotype (Ly-5.1 vs. Ly-5.2) accorded with the RFLP pattern. Cells of the ST/bJ mouse strain have unique Ly-5 serological reactions and ST/bJ DNA gives a unique (third) RFLP pattern (Ly-5c) with pLy-5-68. All Ly-5+ cell types reacted positively with pLy-5-68 in RNA transfer blotting, and all Ly-5- cell types tested did not. The difference in size of mRNA reactive with pLy-5-68 in cells expressing the 200-kDa Ly-5 isoform as compared with cells expressing the 220-kDa Ly-5 isoform corresponded with the difference in size of the protein components of those isoforms.