The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galα1,3-Gal epitope, which is synthesized by α1,3-galactosyltransferase (α1,3-GT). The Galα1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galα1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galα1,3-Gal antigen is the most attractive option. In this study, the 5′ end of the α1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the α1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an α1,3-GT knockout allele and express a randomly inserted human α1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous α1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the α1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.