A small subset of leukemic cells from most patients with acute myeloblastic leukemia (AML) have properties of stem cells and can be assayed by colony formation in agar or methylcellulose. Colony formation generally requires the addition of exogenous growth factors, but the exact factors required are incompletely defined. The AML colony-promoting activities of two recombinant human colony-stimulating factors (GM-CSF and G-CSF) were investigated by using blasts from 48 patients with AML. In nine cases, no colonies formed with either CSF. In seven cases colonies formed only in response to G-CSF and in 11 cases only in response to GM-CSF. In 21 cases colonies formed in response to either GM-CSF or G-CSF, and in 12 of these cases there was an additive effect between the two CSFs in determining maximum colony size. For cases responding to both GM- and G-CSF, the total number of colonies formed in response to the combination of both CSFs was almost always less than additive compared with the number of colonies formed in response to the individual CSFs. Further, the AML-CFU responding to either GM-CSF or G-CSF could not be distinguished by surface markers or by the cytochemical staining pattern of the colonies. These results suggest that there is considerable overlap between the GM-CSF- and G-CSF-responsive AML-CFU subpopulations in most cases. For five of seven cases, the combination of GM-CSF and G-CSF could replace a leukocyte feeder layer in providing maximum growth stimulation. These results indicate that GM-CSF and G-CSF are active growth factors for AML cells and are frequently additive in promoting maximum colony size.