Phycoerythrin (PE)-labeled murine monoclonal antibodies (MAB) to CD2, CD10, CD19, CD20, and CD33 were used as lineage markers, as were PE-labeled anti-DR MAB and fluorescein isothiocyanate (FITC)-conjugated MAB to CD34. One hundred CD34+Lin+DR+ or CD34+Lin-DR- cells were individually sorted and incubated without adherent cell layer in alpha-medium with or without cytokines such as 100 U/mL recombinant human interleukin-3 (rhIL-3), 100 U/mL rhIL-6, and/or 500 ng/mL mast cell growth factor (MGF). The incubated cells were harvested and cultured in medium containing methylcellulose every 2 weeks. The numbers of colonies from colony-forming units-granulocyte/macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and mixed colony-forming units (CFU-Mix) were scored on day 14. In the incubation, CD34+Lin-DR- cells from bone marrow and cord blood produced more CFU-GM and BFU-E, and for longer periods, than did CD34+Lin+DR+ cells. In addition, CD34+Lin-DR- from cord blood supplied more CFU-GM and BFU-E than did CD34+Lin-DR- from bone marrow. Although these data demonstrate that MGF, IL-3, and IL-6 synergistically stimulate the production of CFU-GM and BFU-E, this study indicates that CD34+Lin-DR- cells contain more primitive hematopoietic progenitors and that CD34+Lin-DR- cells are unable to maintain their self-renewal capacity in the presence of IL-3, IL-6, and MGF without adherent cell layer.