Acute myeloid leukemia colony forming cells (AML-CFU) require the addition of colony stimulating factors (CSFs) for in vitro proliferation. Recently, we isolated a human recombinant multilineage CSF (hMulti-CSF). We investigated the ability of hMulti-CSF to stimulate AML clonogenic cells in seven patients in direct comparison with the effects of human granulocyte CSF (hG-CSF), human granulocyte-macrophage CSF (hGM-CSF), and feeder leukocytes. We show that hMulti-CSF is an efficient stimulator of AML colony formation in four of seven cases. In these patients, hGM-CSF was also capable of stimulating AML colonies in vitro. In two of seven cases hMulti-CSF appeared to be a weak stimulus of AML-CFU proliferation. In these latter two cases, however, hG-CSF and in one case hGM-CSF effectively stimulated AML-CFU growth. In one patient none of the hCSFs, either alone or in combination, induced AML colony formation, whereas AML colonies consistently appeared in the phytohemagglutinin (PHA) leukocyte feeder assay. This finding suggests that PHA stimulated leukocytes produce components other than the tested hCSFs that may have a role in the proliferation of AML cells in vitro. Multi-CSF, like hGM-CSF, revealed a limited capacity to induce progressive maturation during AML colony growth, ie, not beyond the promyelocytic stage. On the other hand, in one case, hG-CSF stimulated the growth of AML colonies containing (meta)myelocytes and granulocytes. We conclude that hMulti-CSF is a regulator of AML-CFU proliferation in a significant number of cases. The patterns of responsiveness of AML precursors to the three hCSFs in different patients show a striking variability, which may indicate that AML-CFU are the neoplastic representatives of normal bone marrow progenitors at different stages of maturation and with distinct CSF requirements.