Studies of human and murine bone marrow explants have demonstrated the existence of stromal cell precursors that give rise to colonies of adherent cells in short-term cultures. Because previous data suggested that these colonies were composed of fibroblasts, the precursor cells were termed fibroblast colony-forming units (CFU-F). However, we have recently shown that the stromal cells which support hematopoiesis in murine long-term bone marrow cultures (LTBC) express collagen IV and laminin, markers associated with an endothelial cell lineage, but are negative for collagen I and III, markers associated with a fibroblast cell lineage. Because these conflicting results suggest major functional differences between the stromal cells observed in long-term cultures and the short-term assay, we re-examined the lineage of CFU-F- derived stromal cells. Using two-color immunofluorescence, we characterized virtually all of the cells comprising individual “CFU-F” colonies derived from mouse radiation chimeras. Identification of donor (hematopoietic) or host (stromal) origin was based on surface staining for strain-specific H-2 surface antigens, and, for endothelial or fibroblast properties, on cytoplasmic staining for laminin and collagen IV, or collagens I and III, respectively. The results demonstrate that a large proportion of the cells in CFU-F colonies are donor-derived and fail to stain with any of the antisera specific for nonhematopoietic cells. In addition, these donor-derived cells exhibit marked phagocytic capacity and stain positively with monoclonal antibodies characteristic of the monocyte-macrophage hematopoietic cell lineage (anti-T200, anti- Mac-1, F4/80). However, the remainder of the cells are host-derived cells that stain positively with antisera to collagen IV and laminin. In contrast, stains for collagen types I and III were negative under conditions that allowed for strong staining of control skin fibroblasts. In separate studies, using mixtures of two genetically distinct bone marrows, the cells expressing collagen IV were further shown to be clonal in origin within individual colonies, directly demonstrating that the CFU-F assay provides a quantitative measure of the numbers of marrow stromal cell precursors. Thus, the current studies establish a remarkable similarity between the hematopoietic microenvironment in the short-term CFU-F assay and the long-term culture system: the majority of adherent cells are hematopoietic cells of the monocyte-macrophage lineage, while the remainder are stromal cells whose precise lineage remains uncertain, but whose pattern of collagen expression is more consistent with an endothelial rather than a fibroblast cell origin.