We have previously demonstrated that we could separate long-term repopulating stem cells from cells that provided radioprotection (short-term repopulating cells) on the basis of size and suggested that this might be due to the quiescent nature of long-term repopulating cells. To further define the activity of these populations, we used a dye (PKH26), which incorporates into the membrane of cells and is equally distributed to daughter cells when they divide. We developed an assay, which allowed us to retrieve PKH26(+) long-term and short-term repopulating cells in the hematopoietic tissues of the recipients posttransplant. We were able to recover the labeled cells and determine their cell cycle activity, as well as their ability to reconstitute secondary lethally irradiated hosts in limiting dilution. The results of our assay suggest that long-term repopulating cells are quiescent in the bone marrow (BM) 48 hours after transplant. We were able to detect only a few labeled cells in the peripheral blood posttransplant and even though cells homed to both the spleen and BM, more long-term repopulating cells homed to the marrow and only these cells, which homed to the marrow, were capable of reconstituting lethally irradiated secondary hosts long-term.