Whether hematopoietic stem cells (HSCs) home selectively to bone marrow (BM) early after transplantation remains an issue of debate. Better understanding of homing mechanisms may benefit BM transplantation protocols in cases of limited graft cell number or nonmyeloablative conditioning regimens. Using flow cytometry and serial transplantation to stringently identify HSCs, trafficking patterns of long-term engrafting cells were mapped between BM and spleen early after transplantation. Low-density BM cells were tracked in irradiated or nonirradiated mice 1, 3, 6, and 20 hours after transplantation, at which time recipient BM and spleen were analyzed for recovery of primitive donor cells by phenotype and adhesion molecule expression. In addition, phenotypically defined HSC-enriched or HSC-depleted grafts were tracked 20 hours after transplantation in recipient BM and spleen and analyzed for recovery and long-term repopulating potential in mice undergoing serial transplantation. Regardless of irradiation status, recovery of donor Sca-1+ lin- cells was higher at most time points in recipient BM than in spleen, while recovery of total Sca-1+ cells was variable. A significantly higher percentage of BM-homed donor Sca-1+ cells expressed CD43, CD49e, and CD49d 20 hours after transplantation than spleen-homed cells, which contained significantly more non-HSC phenotypes. Furthermore, BM-homed cells were significantly enriched for cells capable of secondary multilineage hematopoiesis in mice undergoing serial transplantation compared with spleen-homed cells. These results support the notion of specific homing of HSCs to BM by 20 hours after transplantation and provide a basis for the enhanced engraftment potential afforded some Sca-1+ lin- cells subfractionated on the basis of adhesion molecule expression.