Minor histocompatibility (H) Ags elicit T cell responses and thereby cause chronic graft rejection and graft-vs-host disease among MHC identical individuals. Although numerous independent H loci exist in mice of a given MHC haplotype, certain H Ags dominate the immune response and are thus of considerable conceptual and therapeutic importance. To identify these H Ags and their genes, lacZ-inducible CD8+ T cell hybrids were generated by immunizing C57BL/6 (B6) mice with MHC identical BALB.B spleen cells. The cDNA clones encoding the precursor for the antigenic peptide/Kb MHC class I complex were isolated by expression cloning using the BCZ39.84 T cell as a probe. The cDNAs defined a new H locus (termed H60), located on mouse chromosome 10, and encoded a novel protein that contains the naturally processed octapeptide LTFNYRNL (LYL8) presented by the Kb MHC molecule. Southern blot analysis revealed that the H60 locus was polymorphic among the BALB and the B6 strains. However, none of the H60 transcripts expressed in the donor BALB spleen were detected in the host B6 strain. The expression and immunogenicity of the LYL8/Kb complex in BALB.B and CXB recombinant inbred strains strongly suggested that the H60 locus may account for one of the previously described antigenic activity among these strains. The results establish the source of an immunodominant autosomal minor H Ag that, by its differential transcription in the donor vs the host strains, provides a novel peptide/MHC target for host CD8+ T cells.