The effects of three purified colony-stimulating factors (CSFs) with different specificities for the granulocyte (G) and macrophage (M) lineages (G-CSF, CSF-1 and GM-CSF) were studied in a serum-free clonal assay system. The results were compared with those obtained in similar cultures containing fetal calf serum (FCS). Total clone (≥ 10 cells) and colony (≥ 50 cells) numbers were enhanced by FCS under most conditions. However, the extent of enhancement was highly dependent on the concentration and type of CSF. In some instances, FCS also altered the proportions of G, M, and mixed GM clones induced by the CSFs. In cultures stimulated with GM-CSF, enhancement by FCS was significant only at low CSF concentrations, primarily due to increased numbers of M clones. In contrast, clonal growth was increased by FCS only at high concentrations of CSF-1. Clone and colony numbers induced by G-CSF were greatly increased in cultures with FCS at all CSF concentrations tested. Virtually all clones developing in serum-free medium with G-CSF were pure G, whereas, M and GM clones were usually present in serum-containing cultures with high doses of G-CSF. The effects of hydrocortisone (HC) were also examined in these experiments. Like modulation by FCS, modulation of clonal growth by HC depended on the CSF used as stimulus, having no effect in cultures with G-CSF, inhibitory effects with CSF-1, and variable effects with GM-CSF related to CSF concentration.