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Natural killer (NK) cells participate in pig-to-primate xenograft rejection both by antibody-dependent and -independent mechanisms. A majority of human NK cells express the inhibitory receptor CD94/NKG2A, which binds specifically to human leukocyte antigen (HLA)-E, a trimeric complex consisting of the HLA-E heavy chain, β2-microglobulin (β2m), and a peptide derived from the leader sequence of some major histocompatibility complex class I molecules.To use this mechanism for protection of pig tissues against human NK cell-mediated cytotoxicity, we generated transgenic pigs by pronuclear microinjection of genomic fragments of HLA-E with an HLA-B7 signal sequence and of human β2-microglobulin (huβ2m) into zygotes.Three transgenic founder pigs were generated. Northern blot analysis of RNA from peripheral blood mononuclear cells revealed the presence of the expected transcript sizes for both transgenes in two of the three founders. The founder with the highest expression and his offspring were characterized in detail. Fluorescence-activated cell sorting (FACS) and Western blot analyses demonstrated consistent expression of HLA-E and huβ2m in peripheral blood mononuclear cells. Immunohistochemistry revealed the presence of HLA-E and huβ2m on endothelial cells of many organs, including heart and kidney. In vitro studies showed that lymphoblasts and endothelial cells derived from HLA-E/huβ2m transgenic pigs are effectively protected against human NK cell-mediated cytotoxicity, depending on the level of CD94/NKG2A expression on the NK cells. Further, HLA-E/huβ2m expression on porcine endothelial cells inhibited the secretion of interferon (IFN)-γ by co-cultured human NK cells.This novel approach against cell-mediated xenogeneic responses has important implications for the generation of multitransgenic pigs as organ donors for clinical xenotransplantation.