Hyperacute rejection, mediated by natural antibody, is the major barrier to xenotransplantation. The studies reported herein were aimed at evaluating antibody-mediated cytotoxicity and the role of the Gal alpha(1,3)Gal epitope, which we had previously demonstrated was the major epitope of pig cells detected by naturally occurring human antibodies. Also, we had shown that this epitope could be induced in non-expressing cells by the transfection of a cDNA clone encoding alpha(1,3)galactosyl transferase, the enzyme that produces this epitope. The importance of the Gal alpha(1,3)Gal epitope was supported by (1) sugar inhibition studies; (2) complete absorption of cytotoxic antibodies by melibiose-sepharose columns; and (3) the ability of normal human serum to lyse COS cells after transfection with a cDNA clone encoding alpha(1,3)galactosyl transferase. These findings strongly suggest that the majority of cytotoxic human antibodies that would recognize a xenogeneic graft are directed to the Gal alpha(1,3)Gal epitope.