We have studied in detail the effects of cholera toxin (CT), its pentameric B-chain subunit (toxoid) and the A-promoter chain on the differentiation of hemopoietic progenitor cells. Murine marrow cells were treated either with CT or its subunits. After stimulation with either the multilineage growth factor (multi-CSF; also called interleukin 3 or HCGF) or other hemopoietic regulators (colony-stimulating factors, or CSF), the clones grown in semisolid collagen cultures were scored in situ. Pluripotent stem cells (CFU-S) and multilineage (mixed CFU) or lineage-restricted progenitors (CFU-c) were estimated. We found that CT sensitivity is gradually gained by cells through the stepwise differentiation processes (i.e., CFU-S less than multilineage CFC less than committed CFC less than maturing cells). CT also has a selective, dose-dependent inhibitory effect (1 microM to 1 pM) on hemopoietic lineages (basophil-mast cells less than megakaryocytes less than neutrophils less than monomacrophages). These phenomena were obvious when the clonal growth was supported by multi-CSF but, interestingly, were not observed when lineage-restricted CSF were used. They furnished additional evidence that multi-CSF activates cells in a specific manner. This growth factor involved in progenitor cell self-renewal control may contribute to maintaining, on maturing cells, characteristics that are normally the attributes of progenitors.