Murine high proliferative potential colony-forming cells (HPP-CFC) are known to be heterogenous with respect to proliferative capacity and in vitro responsiveness to hematopoietic growth factors. We have separated HPP-CFC into several subpopulations using counterflow centrifugal elutriation. Although HPP-CFC were identified in all of the elutriated fractions of both C3H/HeJ and C57BI/6J bone marrow cells, the distribution of HPP-CFC as well as of colony-forming units-granulocyte- macrophage (CFU-GM) in each fraction differed between these two strains of inbred mice. Six subsets of HPP-CFC were resolved that differed in growth factor responsiveness. A low-density HPP-CFC subpopulation was isolated that was distinct from day-12 spleen colony-forming units (CFU- S12), CFU-GM, and bone marrow stromal cells. This unique subpopulation of HPP-CFC is rate (3% to 9% of total HPP-CFC), appears to be lymphocyte-like in morphology, and behaves the most primitive of the HPP-CFC subsets by requiring multiple hematopoietic growth factors for optimal in vitro cloning. Further characterization of this subpopulation of HPP-CFC will determine the position of these cells in the HPP-CFC heirarchy.