A major problem hampering effective stem cell-based therapies is the absence of a clear understanding of the human hematopoietic stem cell (HSC) pool composition. The severe combined immunodeficiency (SCID) repopulating cell (SRC) xenotransplant assay system provides a powerful tool for characterizing the frequency, cell surface markers, cell cycle status, homing and response to cytokine stimulation of human HSCs. Clonal tracking of retrovirally transduced SRCs and transplantation of specific subpopulations revealed SRC classes with distinct repopulation potentials. However, all HSC repopulation assays are based on intravenous injection, a complex process that requires circulation through blood, recognition and extravasation through bone marrow vasculature, and migration to a supportive microenvironment. Thus, some classes of HSCs may remain undetected. By direct intrafemoral injection, we identified rapid SRCs (R-SRCs) within the Lin-CD34+CD38loCD36- subpopulation. R-SRCs rapidly generate high levels of human myeloid and erythroid cells within the injected femur, migrate to the blood and colonize individual bones of non-obese diabetic (NOD)-SCID mice within 2 weeks after transplantation. Lentivector-mediated clonal analysis of individual R-SRCs revealed heterogeneity in their proliferative and migratory properties. The identification of a new HSC class and an effective intrafemoral assay provide the tools required to develop more effective stem cell-based therapies that rely on rapid reconstitution.