Hemopoietic precursors are heterogeneous with respect to their capacity for self-renewal and long-term repopulating ability. Bone marrow cultures produce a variety of precursors over many weeks, including CFU-S; however, it is important to determine whether these populations retain the functional ability shown by fresh marrow. The most primitive precursor or stem cells have the most long-term repopulating ability. We here describe direct measurements of this ability in cells from marrow cultures by using competitive repopulation assays. Cultured adherent cells repeatedly showed less capacity than fresh marrow cells to repopulate erythropoiesis in irradiated recipients, whereas cultured suspension cells consistently had less capacity than adherent cells. Concentrations of macroscopic CFU-S measured at nine or 12 days were similar in cultured adherent and suspension cells and generally lower than those in fresh marrow. In every experiment, the long-term repopulating ability of the marrow cells used was substantially reduced after transfer into tissue culture. Thus, primitive stem cells may not proliferate in such cultures despite extensive production of CFU-S and more differentiated cell types.