We have previously reported on the successful engraftment and long-term multilineage expression (erythroid, myeloid, lymphoid) of human adult marrow CD34+ HLA-DR- cells in sheep after transplantation in utero. The persistence in these animals of CD34+ HLA-DR- cells, the phenotype of the original graft, and the presence of assayable high proliferative potential colony-forming cells (HPP-CFCs), a primitive progenitor cell that has self-renewal capacity and multilineage differentiative capacity, suggested that these chimeric animals continued to contain human pluripotent hematopoietic stem cells (HSCs). To test this hypothesis, CD45+ cells from the bone marrow of such an animal were isolated by panning at 19 and 24 months after birth, were pooled, and were transplanted into 4 preimmune sheep fetuses (0.6 x 10(6) viable cells per fetus). Three of the sheep were born alive. Bone marrow from one of these secondary recipients (lamb 58) exhibited significant donor (human) cell presence at 3, 9, 12, and 15 months (last evaluation period) after transplant. The engraftment was sustained along with multilineage expression involving lymphoid, erythroid, and myeloid cells of human origin. These results indicated that human CD34+ HLA-DR- cells are capable of long-term marrow repopulation and that this cell population is enriched for human long-term repopulating HSCs.