Human cytomegalovirus encodes several proteins that interfere with expression of major histocompatibility complex (MHC) class I molecules on the surface of infected cells. The unique short protein 2 (US2) binds to many MHC class I allomorphs in the endoplasmic reticulum, preventing cell surface expression of the class I molecule in question. The molecular interactions underlying US2 binding to MHC class I molecules and its allele specificity have not been fully clarified. In the present study, we first compared the sequences and the structures of US2 retained versus non-retained human leukocyte antigen (HLA) class I allomorphs to identify MHC residues of potential importance for US2 binding. On the basis of this analysis, 18 individual HLA-A2 mutants were generated and the ability of full-length US2 to bind wild-type and mutated HLA-A2 complexes was assessed. We demonstrate that Arg181 plays a critical role in US2-mediated inhibition of HLA-A2 cell surface expression. The structural comparison of all known crystal structures of HLA-A2 either alone, or in complex with T cell receptor or the CD8 co-receptor, indicates that binding of US2 to HLA-A2 results in a unique, large conformational change of the side chain of Arg181. However, although the presence of Arg181 seems to be a prerequisite for US2 binding to HLA-A2, it is not sufficient for binding to all MHC class I alleles.