Submyeloblative conditioning regimens for bone marrow transplantation (BMT) are desirable to reduce host toxicity in many settings. We previously demonstrated that hosts conditioned with 160 cGy or with two sequential doses of the antimetabolite 5-fluorouracil (5-FU) displayed ~50% and ~35% donor chimerism 6 months after transplantation of 20 x 106 fresh congenic bone marrow (BM) donor cells, respectively. Donor chimerism in 160 cGy- or 5-FU-conditioned mice was proportional to the reduction in host long-term repopulating ability (LTRA) caused by the conditioning regimen (~80–90% vs. ~55%, respectively). In this study, we sought to determine if donor marrow homing efficiency also correlated with long-term engraftment in these models of submyeloablative conditioning. We hypothesized that sequential doses of the cell cycle-specific agent 5-FU (5-FU x 2) may damage the marrow microenvironment less than low-dose radiation, resulting in more efficient homing in 5-FU x 2-conditioned mice. C57Bl6/J (Bl/6) hosts were conditioned with either 5-FU x 2 administered on days -5 and -1 (150 mg/kg IP) or with 160 cGy given the day of transplantation (day 0). Bl/6 (CD 45.2+) hosts were transplanted with 20 x 106 PKH-26 stained Bl/6 or with Boy J (CD 45.1+) whole BM cells via tail vein injection. The percentage of homed cells 20 hours after BMT in 5-FU x 2-conditioned mice was 3.53 % ± 3.1% (n = 13 from 3 independent experiments), while the percentage of homed cells in the 160 cGy group was 8.67 % ± 2.1% (n = 12; p = 0.0375). The fraction of donor cells which homed to the spleen or remained in the peripheral blood after 20 hours was similar between conditioning regimens. This difference in homing efficiency led us to consider other possible explanations as to why BM cells home less efficiently in 5-FU x 2-conditioned hosts than in low-dose irradiated mice. We hypothesized that 5-FU x 2 may have a more injurious effect on the marrow microenvironment than previously thought. Examination of marrow stromal cell potential using a colony-forming unit-fibroblast (CFU-F) assay revealed that marrow from 5-FU x 2-treated mice (n = 3) contained 1.1 ± 0.4 colonies/marrow femur equivalent (FE), whereas marrow from 160 cGy-treated mice (n = 3) contained 33.8 ± 13.4 colonies/FE. In comparison, marrow from untreated mice (n = 3) averaged 95.6 ± 27.5 colonies/FE. These data suggest that homing efficiency correlates with donor chimerism in these two models of submyeloablative conditioning, and establish at least two potential mechanisms (a lesser reduction of host LTRA, and decreased donor cell homing efficiency) for the lower level of long-term donor chimerism observed in 5-FU x 2- compared to 160 cGy-conditioned hosts. Furthermore, 5-FU x 2 conditioning appears to disrupt the marrow microenvironment to a greater degree than low-dose radiation. Current efforts are aimed at exploring the molecular interactions of homed donor cells with conditioned marrow.