The cell surface expression of HLA-E molecules by transfection is faint in xenogeneic cells. Therefore, this study was done for the aim of better expression of HLA-E molecules on the surface of pig cells in order to overcome xenograft rejection mediated by human natural killer (NK) cells. The importance of the loading peptide sequence for HLA-E expression has been studied extensively, but much less information is available concerning the HLA-E heavy chain sequence. In our previous study, we developed the S147C substitution of HLA-E as a useful gene tool for xenotransplantation. In this study, a more extensive substitution analysis throughout the entire region led to the identification of nine amino acid positions, positions-9, 11, 25, 40, 66, 67, 74, 99 and 174, that are significantly involved in the cell surface expression of HLA-E molecules. In view of xenotransplantation usage, double and triple point substitutions, HLA-Ev(11,147) and HLA-Ev(11,66,147), were constructed. These constructs led to a high expression on the xenogeneic cell surface and possessed inhibitory functions against human NK cell-mediated cytolysis in an in vitro pig to human xenotransplantation model system.