Background.Animal cloning techniques have enabled gene disruption in several species. Here, we report the first successful disruption of the &agr;1,3-galactosyltransferase (&agr;1,3-GT) gene in cattle.Methods.The &agr;1,3-GTgene of the Japanese Black cow (JBC) was used to construct pGT-6, a targeting vector for the bovine &agr;1,3-GTgene, and pGT-6 was introduced into the fetal fibroblast cell line JBC906 by the lipofection method. Four polymerase chain reaction (PCR)-positive colonies were obtained from 797 G418-resistant colonies, and Southern blot analysis revealed successful homologous recombination at the &agr;1,3-GTlocus in one of the four colonies. Nuclear transfer was performed, and the four embryos were transferred to a heifer.Results.To establish fetal fibroblasts that were heterozygously disrupted at the &agr;1,3-GTlocus, one of the fetuses was recovered at 5 weeks of pregnancy, and PCR and Southern blot analysis of the fetal fibroblasts established from it showed definite homologous recombination of the &agr;1,3-GTgene.Conclusions.Heterozygous knockout of the &agr;1,3-GTgene was performed in JBC, and production of a homozygous &agr;1,3-GTknockout JBC by a second round of targeting 906htGT is currently in progress. The technique described here can be applied to disruption of other genes in cattle.