The effects of colony-stimulating factors on differentiation of leukemic progenitor cells were investigated in various types of acute nonlymphocytic leukemia. Two different sources of colony-stimulating factors were used in this study: human placental conditioned medium; and phytohemagglutin-stimulated leukocyte-conditioned medium. At the end of culture, colony types were determined by dual esterase staining in permanent preparations. The majority of the colonies formed in acute myeloblastic and acute promyelocytic leukemias were neutrophilic even when stimulated by phytohemagglutinin-stimulated leukocyte-conditioned medium, which contains a potent stimulator of macrophage colony growth from normal marrow cells. On the other hand, both neutrophilic and monocytic colonies were formed in acute myelomonocytic leukemia (AMMoL). The proportions of these two types of colonies were variable, depending on the nature of added colony-stimulating factor and its concentration. These findings suggest that the leukemic progenitors in acute myeloblastic and acute promyelocytic leukemias have a tendency to differentiate mainly into a neutrophilic lineage in vitro and that the leukemic progenitors in AMMoL differentiate into both neutrophilic and monocytic lineages in vitro, in two cases of esterase-negative AMMoL, both neutrophilic and monocytic colonies were detected as in the other well-defined cases of AMMoL. This study seems to be of value in understanding the nature of leukemic progenitor cells and also shows that morphological analysis of leukemic colonies may be helpful in the classification of acute nonlymphocytic leukemia.