Recombinant (r) and natural human (h) macrophage colony-stimulating factor (M-CSF, CSF-1) have been considered poor stimulators of macrophage progenitor cells present in human marrow, although they are potent stimulators of these cells in mouse marrow. We compared the growth characteristics of rhM-CSF-responsive human macrophage progenitor cells placed in semisolid agarose or agar culture medium and incubated for 14 days at ambient (approximately 20%) or lowered (5%) O2 tension. By itself, rhM-CSF was found to be a good stimulator of macrophage colony formation by human bone marrow cells cultured in agarose but not in agar; this growth was enhanced by incubation at 5% O2. Maximal numbers (up to 115/10(5) nonadherent low density cells plated) of macrophage colonies (50 to greater than 500 cells per colony) were stimulated by 500 to 1,000 units rhM-CSF/mL, with 1/2 maximal numbers stimulated by 250 to 500 units/mL. With agarose as the support medium, rhM-CSF was two- to fourfold more active on mouse than on human macrophage colony formation, in contrast to previous reports of 10- to 100-fold greater activity when agar was used as the support medium. Using nonadherent low density T lymphocyte-depleted human bone marrow cells growing in agarose at 5% O2, greater than additive effects on colony formation were observed when 31 to 500 units rhM-CSF were used in combination with either 10 ng rh interleukin-1 alpha (IL-1 alpha), 20, or 200 units rh granulocyte-macrophage (GM)-CSF or rhG-CSF. The agarose assay system should be useful for evaluating factors regulating the proliferation of human macrophage progenitor cells in vitro and during clinical trials with rhM-CSF.