Autocrine growth mechanisms of leukemic blast progenitors in acute myeloblastic leukemia (AML) were investigated. Colony formation of leukemic blast progenitors was observed in 14 of 14 patients tested when purified blast cell fraction depleted of both T cells and monocytes was plated in methylcellulose without any colony-stimulating factor (CSF). However, there existed a minimal cell density required to initiate blast progenitor growth with marked patient-to-patient variation. To clarify the role of cell density on the spontaneous growth of blast progenitors, we tested whether leukemic cells produced and secreted some stimulatory humoral factor(s). Production of colony-stimulating activity (CSA) by blast cells was observed in 17 of 18 patients tested. Following further depletion of monocytes, the CSA levels decreased markedly in 14 patients, indicating that blast cells with monocytoid differentiation were responsible for CSA production. We also confirmed granulocyte colony-stimulating factor (G-CSF) and/or granulocyte macrophage-colony-stimulating factor (GM-CSF) production by leukemic blasts using specific immunologic assays. When leukemic cells were divided into nonadherent nonphagocytic cell fraction and adherent cell fraction, only nonadherent nonphagocytic cells showed clonogenecity and adherent blast cells lacked the colony-forming capacity. The results indicate that there are at least two blast cell subpopulations in AML: one is proliferating subpopulation with self-renewal capacity and the other is supporting subpopulation with functions such as CSF production. The quite intimate relationship between these two blast cell subpopulations in AML may play an important role on the growth of leukemic blast progenitors in vitro.