Highly enriched, phenotypically defined hematopoietic stem, Thy-1loLin-Sca-1+, and progenitor cell populations from mouse bone marrow (BM) were tested at limiting dilution for their ability to reconstitute Dexter monolayers. Several classes of BM cells can reconstitute Dexter cultures, first forming discrete "cobblestone" areas which then mature into colonies consisting primarily of maturing myeloid and erythroid cells. Most such colonies have a limited lifespan in culture. Only the Thy-1loLin-Sca-1+ cell fraction gives rise to colonies that survive longer than 3 weeks, which suggests that a limiting-dilution analysis for long-term reconstitution of Dexter cultures can serve as a quantitative measure of stem cell activity. Additional experiments were performed to assess the formation of new progenitor cells in reconstituted Dexter cultures. Again, only cultures seeded with the stem cell-enriched fraction contained expanded numbers of replatable WEHI-3 CM responsive colony-forming cells (CFU-GM). Quantitative analysis indicates that 97% of the replatable CFU-GM of whole BM is contributed by the Thy-1loLin-Sca-1+ cell fraction, again suggesting a potential stem cell-specific assay. Such quantitative in vitro assays might prove useful in characterization and isolation of human stem cells where in vivo assays are lacking.