This report presents the results of studies investigating the effect of insulin-like growth factor II (IGF-II) on the proliferation and differentiation of CD34+ bone marrow cells in serum-substituted liquid cultures. Bone marrow cells were enriched for CD34+ cells and then placed in liquid cultures supplemented with either interleukin 3 (IL-3) or IL-3 and c-kit ligand with and without the addition of IGF-II. When CD34+ cells were incubated with IL-3, cellularity increased throughout four weeks of culture. Cellularity was twofold greater when cultures also contained IGF-II. IGF-II also promoted an increase in cellularity in cultures with IL-3 and c-kit ligand. In combination with IL-3 or IL-3 and c-kit ligand, IGF-II promoted an earlier differentiation of granulocytes, as well as an increase in the number of megakaryocyte lineage cells. There were approximately two-fold more colony-forming units for granulocytes and macrophages (CFU-GM) and burst-forming units for erythroid cells (BFU-E) in cultures containing both IL-3 and IGF-II than in cultures with IL-3 alone. These results demonstrate that in cytokine-supplemented media, physiological concentrations of IGF-II augmented both the proliferation and differentiation of CD34+ bone marrow cells while maintaining a greater number of progenitor cells. To identify the receptors through which IGF-II enhances in vitro hematopoiesis, IGF-II was substituted with one of the mutant forms of IGF-II that selectively interacts with either IGF-II/CIM6-P receptors or with IGF-I and insulin receptors. The results with the mutant forms of IGF-II demonstrate that IGF-II augments in vitro hematopoiesis primarily through its interaction with IGF-I and possibly insulin receptors, rather than IGF-II/CIM6-P receptors.