Bone marrow or blood of patients with acute myeloid leukemia was subjected to cell separation and the cells investigated for in vitro colony growth. Discontinuous albumin density gradient centrifugation and depletion of E-rosette-forming cells resulted in purified fractions of acute myeloid leukemia cells. From these fractions, growth of large leukemic colonies was obtained in the PHA-leukocyte feeder (PHA-LF) colony technique in 12 of 14 patients. The standard double agar layer techniques with a leukocyte feeder for granulocyte-macrophage colony forming cells (GM-CFC) supported colony formation in only four cases. The PHA-LF leukemic colony-forming cells (CFC) were found to be of low buoyant density (always less than or equal to 1.062 g.ml-1) when compared to normal marrow GM-CFC (peak at 1.065 g.ml-1). The density profile of PHA-LF CFC paralleled the distribution of the nucleated cells in 8 cases, but in 4 patients, the cFC peak was found at a distinctly lower density; this suggested that a specific leukemic subpopulation had a colony-forming capacity. In three of the four patients with colony growth in the double layer agar technique, it was evident that these CFC had density properties different from those of PHA-LF CFC. These findings suggest that cells giving rise to large colonies in the PHA-LF and double layer agar assays represent distinct leukemic subpopulations.