Murine fetal liver (FL) and adult bone marrow (BM) hematopoietic stem cells (HSCs) are characterized by cell surface expression of CD38 and c-kit. Because murine yolk sac (YS) HSC activity precedes the initiation of FL hematopoiesis, we investigated whether YS-derived HSCs also expressed c-kit and CD38. c-Kit+ CD38+ lineage- cells derived from day 9 YS as well as adult BM were found to be enriched in high proliferative potential colony-forming cells. c-Kit+ CD38+ lineage- YS or adult BM cells were capable of long-term reconstitution (>6 months) of busulfan-conditioned newborn or lethally irradiated adult mice, respectively. In contrast, c-kit+ CD38- lineage- populations from both tissues were enriched in lineage-committed progenitors and had no long-term HSC activity. We concluded that c-kit and CD38 are cell surface markers of HSCs expressed throughout murine ontogeny.