Although activating mutations in ras genes are the most common genetic abnormality in human hematologic malignancies, the role of ras mutations as an initiating event in leukemogenesis remains unclear. To assess the consequences of ectopic expression of an activated ras gene in normal hematopoietic cells in vivo, lethally irradiated mice were reconstituted with bone marrow cells infected with a mutant ras-containing retrovirus [murine stem cell virus (MSCV)-vH-ras] based on the MSCV retroviral vector which efficiently transduces functional genes into hematopoietic stem/progenitor cells. Despite a marked myeloid leukocytosis detectable in the peripheral blood within 4 weeks of engraftment, none of 22 primary or secondary transplant recipients studied for longer periods of time presented with myeloid neoplasms. Instead, 18 of the MSCV-vH-ras mice developed pre-T-cell thymic lymphomas and/or pre-B-cell lymphoblastic leukemia/lymphomas between 7 and 12 weeks post-transplantation. The pre-B and pre-T lymphoid tumors that arose in one animal were shown to harbor a common MSCV-vH-ras provirus, indicating that the target cell for transformation was a bipotential lymphoid precursor. To more precisely examine the effects of activated ras expression on the behavior of hematopoietic progenitors, infected bone marrow cells were assayed in methylcellulose cultures under conditions favorable for growth of multilineage myeloid colonies or were passaged as bulk suspension cultures in the presence of various hematopoietic growth factors, including interleukin (IL)-3, IL-4, IL-6 and IL-7. MSCV-directed expression of vH-ras selectively promoted the formation of large dense colonies comprised of monocyte-macrophages in methylcellulose cultures. When transferred to liquid cultures, the vast majority of the cells underwent terminal macrophage differentiation. By comparison, tumorigenic B-lymphoid and mixed lymphoid/myeloid cell lines were routinely established from the bulk suspension cultures, with cell lines of predominantly myeloid phenotype emerging only in IL-6-supplemented cultures. These results, considered together with previous findings, suggest that activating ras mutations could be an initiating genetic alteration in human acute lymphoblastic leukemia but are more likely to be a post-initiation change in human acute myeloid leukemia.