Xenogeneic transplantation is a potential solution to the severe shortage of donor organs for clinical transplantation. The primary limitation to xenogeneic transplantation between widely disparate species is hyperacute rejection, which is triggered by the recipient's natural antibodies reacting against the donor's endothelial cells. Natural antibodies react between widely disparate species but do not react between closely related species. Specific tolerance for xenogeneic transplantation between closely related species can be induced by creating hematopoietic chimerism between donor and recipient. However, whether specific xenograft tolerance can be induced by the creation of chimerism between widely disparate species, where natural antibody reacts, is unknown. We previously have established a model of hematopoietic chimerism between widely disparate species by the in utero transplantation of human fetal hematopoietic stem cells into early gestation fetal lambs. In the present study, we determined whether long-standing hematopoietic chimerism in this human/sheep model reduces the level of natural antibody directed against human endothelial cells. To answer this question, we measured the reactivity of serum from five chimeric sheep, five sheep controls, and five human controls in an in vitro enzyme-linked immunoabsorbance assay directed against human umbilical vein endothelial cells. Unexpectedly, we found that long-standing hematopoietic chimerism in the human/sheep model did not reduce the reactivity of serum against human endothelial cells compared to age-matched sheep controls. These results suggest that the induction of hematopoietic chimerism between widely disparate species will not control the problem of natural antibody and hyperacute rejection.