We studied the effects of recombinant human interleukin-4 (IL-4) on colony formation by enriched hematopoietic progenitors. IL-4 alone did not support colony formation at all. When IL-4 was combined with granulocyte colony-stimulating factor (G-CSF), the number of pure neutrophil colonies increased three times over that supported by G-CSF alone. IL-4 added 5 days after the addition of G-CSF failed to exert this synergistic effect, indicating that IL-4 acts on the early stage of proliferation. The mapping experiments (sequential observation of colony formation) have clearly shown that IL-4 did not initiate progenitor cell proliferation. Based on these data, IL-4 may possess a direct action on progenitor cells; however, it can only act as a costimulant with G-CSF. In contrast, IL-4 had possible inhibitory effects on macrophage colony formation supported by interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF). In other words, IL-4 may induce progenitor cells to become sensitive to G-CSF and thereby induce neutrophil differentiation. Delayed addition experiments demonstrated that human IL-4, unlike murine IL-4, could support neither proliferation nor survival of erythroid burst or mixed colony forming cells. Neutrophil colony forming cells only survived and recovered after addition of G-CSF and erythropoietin on day 5 of incubation. On the other hand, IL-3 supported neutrophil, erythroid burst, and mixed colony forming cells as reported previously (Sonoda et al, Proc Natl Acad Sci USA, 85:4360, 1988). These results led us to propose that IL-4 possibly acts with more mature progenitor cells than those of IL-3 or granulocyte-macrophage (GM)-CSF.