In order for xenotransplantation to become a clinical reality, and fulfill its promise of overcoming shortages of human organs and tissues, rejection mediated by the host's immune system must first be overcome. In primates, preformed natural antibodies that bind the carbohydrate antigen Galalpha1-3Galbeta1-4GIcNAc-R (alphaGal), which is synthesized by UDP galactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha(1-3)galactosyltransferase (E.C. 2.4.1.151) or simply alphaGT, mediate rigorous rejection of transplanted pig organs and tissues. In alphaGT knockout mice (GT0 mice), which like humans contain in their serum antibodies that bind alphaGal, expression of a retrovirally transduced alphaGT in bone marrow-derived cells is sufficient to prevent production of alphaGal-reactive antibodies. Here, we demonstrate that reconstitution of lethally irradiated GT0 mice with alphaGT-transduced bone marrow cells from GT0 littermates prevents antibody-mediated rejection of cardiac transplants from wild-type mice. These data suggest that gene therapy can be used to induce immunological tolerance to defined antigens and thereby overcome transplant rejection.