PKH-26 was used as a viable fluorescent membrane stain for murine hematopoietic stem cells. The presence of the dye on the cells was shown not to interfere with their ability to form day-8 and-12 spleen colonies in lethally irradiated mice. To study their in vivo homing behavior in detail, 10 (4) labeled cells from a population enriched for CFU-S were injected intravenously into nonirradiated mice and into mice irradiated 3 hours previously. At 17, 41, and 65 hours after injection, the numbers of labeled cells per organ were quantified using the specialty developed flow cytometric fluorescence hypercompensation procedure for the detection of rare events, which allows a detection sensitivity of 1 per 10 (6). Spleen homing in irradiated and nonirradiated mice was virtually identical, whereas homing to nonirradiated bone marrow was 2.5 times higher than to irradiated bone marrow. This indicates a different homing mechanism for spleen and bone marrow. The results of this direct homing assay were placed in perspective with results of indirect homing studies from the literature, introducing a new" h-factor." From the CFU-S data, putative specific enrichment factors for spleen-specific and bone marrow-specific homing were derived. Examination of the fluorescence intensity distribution among the labeled cell population indicated that virtually all cells started to proliferate rapidly after injection into both irradiated and nonirradiated animals. This indicates that specific signals from stromal elements in the stem cell niches are needed to keep the cells quiescent and that the majority of the transplanted stem cells do not home to such niches. The potential use of PKH-26 for in vivo characterization of stem cell niches is discussed.