We have demonstrated previously that interferon-γ (IFN-γ) accelerates platelet recovery in mice with 5-FU induced-marrow aplasia in vivo. However, the mechanism for the regulation of megakaryocyte development induced by IFN-γ in bone marrow cells in vivo remains unknown. To further study the effects of IFN-γ on megakaryocyte development, various steps during IFN-γ-mediated accelerated differentiation of the megakaryocytes were investigated in serum-free cultures of murine bone marrow cells in vitro. IFN-γ markedly induced acetylcholine esterase (AChE) activity, a marker of murine megakaryocytic cells, accompanied by increased colony formation of the megakaryocyte lineage. A prominent increase in megakaryocyte number was observed after IFN-γ treatment. All of these effects were dependent on the presence of IL-3, and, therefore, these results suggest that IFN-γ acts as a megakaryocyte potentiator (Meg-POT). However, IFN-γ did not enhance megakaryocyte maturation with respect to increase in cell size. The effects of IFN-γ on megakaryocyte maturation were similar to those observed after treatment with higher doses of IL-3 alone. Meg-POT is defined as a factor that induces megakaryocyte maturation. Since IFN-γ enhanced IL-3-dependent megakaryocyte colony formation and proliferation rather than megakaryocyte maturation, the effects on megakaryocyte development, which were induced by IFN-γ treatment, seem to be different from the effects of a Meg-POT. We, therefore, propose a new function for IFN-γ as an enhancer of megakaryocyte colony-stimulating factor activity. The effect of IFN-γ in vitro appears to correlate well with the acceleration of platelet recovery in vivo.