We performed a subpopulation analysis in acute myelocytic leukemia (AML) with the objective of relating the phenotypic diversity to the clonogenic properties of the cells. AML-CFUs considered as representatives of the leukemia precursor cell compartment were phenotyped for cell surface markers by use of eight monoclonal antibodies directed against myeloid differentiation antigens and the Ia antigen. Thus, specifically depleted (in complement-dependent lysis) or selected (in fluorescence-activated cell sorting), cells were inoculated into colony culture. We report here that AML-CFU surface phenotypes showed considerable variability among the seven patients with AML studied. The clonogenic cells were heterogeneous with regard to the antigen density on the cell surface and represented a distinct population among the leukemias. The immunophenotypes of AML-CFU did not differ as a consequence of variations of colony techniques, ie, the use of different stimulatory materials. The marrow and blood, as shown in four subjects, contained AML-CFU of identical maturation stages. In addition, the direct cellular offspring of AML-CFUs were phenotyped. This was done by analyzing the colonies produced in culture, and it was apparent that their antigenic markers were compatible with later stages of differentiation. For example, AML-CFUs in all patients were granulocytic antigen B4.3-negative, whereas their colony progeny contained significant numbers of B4.3-positive cells. This direct evidence indicating that progenitors and descendant cells in AML are identified by distinct maturation features corroborates the concept that the heterogeneous cellular structure of human AML is a reflection of the apparent differentiation capabilities of the precursors. The fact that the differentiation markers of the leukemia are variably expressed or even lacking on AML-CFUs has clinical importance in the immunodiagnosis of residual leukemia and in immunoseparation of leukemia from autologous bone marrow grafts.