We studied the effects of recombinant human interleukin-4 (rhIL-4) on megakaryocyte colony formation from enriched hematopoietic progenitors. IL-4 strongly inhibited pure and mixed megakaryocyte colony formation in a dose-dependent manner. Formation of erythroid bursts, eosinophil colonies, and erythrocyte-containing mixed colonies was not affected by the addition of IL-4 as reported previously (Sonoda Y, et al; Blood 75:1615, 1990). Delayed addition experiments suggested that IL-4 acts on an early stage of proliferation of megakaryocyte progenitors. Neutralizing antibodies (antisera) prepared against transforming growth factor beta, tumor necrosis factor alpha, interferon alpha (IFN alpha), and IFN gamma did not affect the inhibitory effects of IL-4 on pure and mixed megakaryocyte colony formation. In addition, the inhibitory effects of IL-4 was also seen in serum-free cultures and in cultures containing highly enriched CD34+, HLA-DR+ cells as a target population. These results indicate that IL-4 may function as one of the negative regulators in human megakaryocytopoiesis in vitro.