Three different HLA-A2.1 monochains were engineered in which either the human or mouse β2-microglobulin (β2m) is covalently linked to the NH₂ terminus of the heavy chain by a 15– amino acid long peptide: HHH, entirely human, HHD, with the mouse H-2D^b α3, transmembrane, and cytoplasmic domains, and MHD, homologous to HHD but linked to the mouse β2m^b. The cell surface expression and immunological capacities of the three monochains were compared with transfected cells, and the selected HHD construct was introduced by transgenesis in H-2D^b−/− β2m^−/− double knockout mice. Expression of this monochain restores a sizable peripheral CD8⁺ T cell repertoire essentially educated on the transgenic human molecule. Consequently, infected HHD, H-2D^b−/− β2m^−/− mice generate only HLA-A2.1–restricted CD8⁺ CTL responses against influenza A and vaccinia viruses. Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1–restricted immunodominant epitope in humans. Such mice might constitute a versatile animal model for the study of HLA-A2.1–restricted CTL responses of vaccine interest.