The HLA-DO molecule, a heterodimer consisting of two novel members of the class II gene family, DOA and DOB, has recently been suggested to function as an important modulator in the HLA class II restricted antigen presentation pathway by interaction with the HLA-DM molecule. In this study, we have analyzed genetic polymorphism and allelic variation of the HLA-DOA gene in 37 HLA class II homozygous typing cells using the direct DNA sequencing technique. As a result, we recognized at least eight allelic variations, DOA*01011, *0101201, *0101202, *0101203, *01013, *0101401, *0101402 and *01015. None of them, however, result in amino acid substitution. The HLA-DOA gene has been identified in other mammals as well, and the nucleotide sequences were well conserved among these species. These results suggest that the DOA molecule has undergone strong selective pressure to preserve functional structure and conformation required for interaction with the DM molecule, preventing non-synonymous amino acid substitution.