Three cDNA with D region gene features have been identified from the H-2v haplotype. Provisionally, the sequences have been designated as D/Lv1, D/Lv2, and D/Lv3. The coding segments for the antigen binding domain (ABD) of all three D/Lv genes were engineered into a class I genomic expression vector and expressed in L cells. FACS analysis of the three D/Lv-Ld gene transfectants revealed that the D/Lv1 molecules were recognized by both monoclonal antibodies (mAbs) 141 and 142, and the D/Lv2 molecules were recognized by mAb 143. In addition to the D/Lv1 molecules, the mAb 141 also recognized the D/Lv3 molecules. Both the D/Lv1-Ld and D/Lv2-Ld transfectants were killed efficiently by H-2Dv region-specific alloreactive CTL. The D/Lv3 gene is the first identified D region gene other than D and L that is transcribed abundantly in spleen and the D/Lv3 RNA is present as two alternatively spliced forms. Structural analysis of the D/Lv3 hybrid molecules showed that it was susceptible to proteolysis and thermolabile at 37 degrees C, suggesting D/Lv3 is a transcribed pseudogene. A parsimony tree analysis of three D/Lv sequences with a set of class I gene sequences revealed that the H-2v sequences clustered with D region genes. The presence of a third gene with D/L-like features in H-2v, yet structurally different from the known D/L alleles, raises the possibility that the current D/L genes evolved from a family of D/L-like genes, some of which are no longer represented among many of the mouse major histocompatibility complex haplotypes. The observation that D region alleles cluster into subgroups suggests that the alleles are not all related to each other by linear descent through a single locus. We propose that current alleles are derived from more than one ancestral locus in a manner similar to the origin of the gamma 2 a immunoglobulin constant region alleles.