To better understand the limited hematopoietic life span of human marrow “Dexter” cultures, we developed a miniaturized, two-stage culture system with which in vitro production of hematopoietic progenitors could be reproducibly detected and quantified. Light- density, gradient-separated human marrow cells were inoculated into Leighton slide tubes, and adherent (“stromal”) cell layers were allowed to develop on the removable coverslips within these tubes during an initial 4 weeks of culture. Once stromal cell layers were established, cultures were irradiated (800 cGy) to eliminate all residual hematopoietic progenitors. The cultures were then recharged with autologous, cryopreserved marrow cells (enriched for BFU-E and CFU-GM) to reconstitute stem cell populations and to initiate in vitro hematopoiesis. Most progenitor cells added to irradiated cultures were no longer detectable by clonal assays within one to four days after recharge. Nonetheless, stable populations of adherent BFU-E and CFU-GM became established in these cultures within 24 to 48 hours, and when the total numbers of progenitors (adherent and nonadherent) were measured at weekly intervals thereafter, it was evident that both BFU-E and CFU-GM were generated in vitro. However, progenitor cell production declined as neutrophils and macrophages accumulated in the cultures. Moreover, with this accumulation of mature myeloid cells, increasing levels of O2- and H2O2 could be detected in the cultures, and it was found that the addition of oxidant scavengers (catalase and mannitol) to culture media enhanced the weekly expansions of progenitor cell numbers that could be measured. These findings support the conclusion that reactive O2 intermediates generated by mature myeloid cells have a role in limiting the duration and extent of hematopoietic progenitor cell self-renewal in long-term “Dexter” cultures of human marrow.