The in vitro study of stem-cell-stromal-cell interactions has previously been made possible by the existence of a technique for long-term murine marrow culture, but the occurrence of both stem cells and stromal cells in fresh bone marrow (BM) explants and the heterogeneity of stromal cells have delayed functional categorization. Therefore, single-cell suspensions of murine BM were passed over nylon wool columns to allow fractionation of cells having distinctive function for in vitro hemopoiesis. A subpopulation of nylon-column-nonadherent (NNA) cells (20% +/- 1% total cells) is devoid of stromal progenitors that form the in vitro microenvironment, but the NNA subpopulation has control numbers of hemopoietic colony-forming cells, GM-CFU-C, and high-proliferative-potential colony forming cells (HPP-CFC). This subpopulation also produces control numbers of in vivo spleen colony-forming cells, CFU-S, and has control numbers of primitive, noncycling colony-forming cells that resist in vitro treatment with 5-fluorouracil (5-FU). By contrast, nylon-adherent populations, when eluted by mechanical agitation (MA) (41% +/- 1%) or by subsequent EDTA treatment (CA) (6% +/- 1%) could reform the hemopoietic microenvironment in vitro. Stromal progenitor cells were negative for surface markers Thy-1 and Mac-1. When NNA stem cells were added to culture with nylon-adherent stromal fractions, lodgement of stem cells occurred, resulting in stem cell proliferation for up to three months.