Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34⁺ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34⁺ Kit^-, CD34⁺ Kit^low, and CD34⁺ Kit^high cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit^low cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow ceils and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34⁺ Kit^high cells and two fetuses receiving CD34⁺ Kit^- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34⁺ Kit^low cells exhibited signs of engraftment upon serial examination. Earlier, in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit^low population.