Background. The major antigen recognized on pig tissue by primate antibodies is a terminal galα1-3gal carbohydrate structure (gal antigen) present on glycolipids and glycoproteins. The production of animals from somatic cells allows for the inactivation of specific genes. It is anticipated that the complete inactivation of the gene encoding α1-3 galactosyltransferase, the enzyme that synthesizes the galα1-3gal linkage, will result in loss of that antigen from pig organs and tissue and will provide a survival benefit in pig-to-primate xenotransplants. Methods. Positive-negative selection was used to produce fetal-pig fibroblasts that were a heterozygous knockout (+/-) of the α1-3 galactosyltransferase gene. Nuclear transfer of these cells generated pig embryos and live born pigs with the appropriate genotype. Using a novel selection method with cells from (+/-) embryos, we produced homozygous (-/-) fetal-pig fibroblast cells. Results. Southern blot analysis of the α1-3 galactosyltransferase gene showed that we had produced (+/-) pig embryos, (+/-) live born pigs, and (-/-) pig-fetal fibroblast cells. Fluorescence-activated cell sorter (FACS) analysis with some, but not all, mouse anti-gal monoclonal antibodies and sensitized human serum showed that (-/-) cells still synthesized the gal antigen at 1 to 2% of the level of control heterozygous cells. Conclusions. Fetal-pig fibroblasts homozygous for the knockout of the α1-3 galactosyltransferase gene appear to express low but detectable levels of the gal antigen.