Poor bone marrow (BM) engraftment in a xenogeneic combination results at least in part from the limited engraftment capacity of BM-derived stromal cells, which support hematopoietic repopulation in a species-specific fashion. We attempted to construct a BM stromal microenvironment by engraftment of BM plug fragments into kidney capsules in a rat-to-mouse combination. BM plugs from F344/N Jcl-rnu/rnu (F344 nu) rats were transplanted into the kidney capsules of C.B-17 scid/scid (C.B-17 scid) mice treated with rabbit anti-asialo-GM1 serum to deplete natural killer (NK) cells and then with 3 Gy of whole body irradiation. As a conventional control, an equivalent amount of F344 nu bone marrow cells (BMCs) was intravenously injected into C.B-17 scid mice treated with a similar conditioning regimen. In both mouse recipients of rat BM plug engraftment in the kidney capsules and recipients of intravenous injection of rat BMC suspension, comparable extents of donor rat class I+ cells were persistently detected in the peripheral blood. However, the differentiation of rat-derived B cells in the mouse recipients of rat BM plugs was more rapid than that in the recipients of rat BMC suspension. In the late phase (10 weeks after BM transplantation), the percentage of rat-derived T cells (CD4+ cells) in the mouse recipients of rat BM plugs was significantly higher than that in the recipients of rat BMC suspension. At this time point, ectopic BM structure consisting of bone, mesenchymal cells, and hematopoietic progenitors was constructed in the kidney capsules of mice that received rat BM plugs. Most of the cells in the ectopic BM were derived from the donor rat. Thus, engraftment of BM plugs into the kidney capsules results in the construction of a donor-derived BM microenvironment, facilitating multilineage mixed xenogeneic chimerism.