Stromal cell lines were established from canine long-term marrow cultures, cloned by limiting dilution, and maintained in stromal cell-conditioned medium. These cells grew adherent, maintained stable growth rate and morphology under standard conditions (in 20-30% conditioned medium; confluency, 70-90%), and supported hemopoiesis in long-term marrow cultures. In the presence of exogenous recombinant canine stem cell factor (rcSCF), round cells developed from the adherent layer, detached, and remained in culture as viable floating cells. Round floating cells also appeared when cultures were grown to > 90% confluency without rcSCF. Round cells were smaller than adherent cells, expressed CD34, showed basophilic plasma, and stained positive for c-kit, MHC-class II markers, and myeloid markers. In standard assays for colony formation, the detached cells produced granulocyte-macrophage colony-forming units (CFU-GM), fibroblast colony-forming units (CFU-F), and less well-defined colony-forming units. In addition, on allogeneic feeder cells in long-term cultures, these cells generated hemopoietic colonies. Strikingly, the differentiation was reversible: when nonadherent cells were resuspended at lower density in serum-containing medium, they reattached and grew to confluence when, once again, round cells detached. Detached cells from this secondary cycle produced mainly CFU-F and few CFU-GM when placed in clonal assays. These results suggest that some fibroblast-like stromal cells have the potential to differentiate into cells with hemopoietic characteristics. These observations provide evidence for the existence of a quiescent precursor of hemopoietic progenitors in the bone marrow stroma of the adult dog.