Two phenomena appear to distinguish the D region class I genes from those in the K region in the murine MHC: (a) haplotype disparity in the number of expressed D region class I molecules has been observed; and (b) clines of closely related D region class I molecules among and within mice of different H-2 haplotypes can be defined. Both of these observations have been based on serological and peptide mapping analyses of these molecules. Recent reports using molecular biological approaches have corroborated these findings. Since the mouse strain B10.AKM expresses multiple D region class I antigens, all of which are closely related to the prototypic Ld molecule, we investigated the Dq region of B10.AKM using molecular approaches. Three D region class I genes were isolated from genomic B10.AKM bacteriophage and cosmid libraries. Based on alignment of those genes with the BALB/c D region class I genes by analogous restriction endonuclease sites and by hybridization of one of those genes with a D4d gene-derived oligonucleotide probe, we have designated these genes as Dq, Lq, and D4q. As determined by DNA-mediated gene transfer to mouse L cells followed by serological analyses, the Dq and Lq genes encode previously characterized Dq region class I antigens. The nucleic acid sequence comparisons of the Dq and Lq genes demonstrated a higher level of homology with the Ld and Db genes than with other D region class I genes. In addition, CTL stimulated with a Dq, Lq, or Ld gene transfectant showed strong crossreactions with the other transfectants as targets, suggesting that the products of these genes are also functionally related. Thus, these studies suggest that the L molecule represents a prototypic structure shared by several D region gene products, and furthermore, the duplication of an Ld-like progenitor gene resulted in two Dq region class I genes, Dq and Lq. Unexpectedly, the sequences determined for the Dq and Lq genes are nearly identical to the sequences of two genes, A166 and A149, respectively, which were reported to encode the tumor-specific antigens; these novel class I genes were isolated from an H-2k fibrosarcoma, 1591. This raises the distinct possibility that these purported tumor-specific class I genes were introduced into this tumor by contamination.