Interferons (IFN) have been shown to suppress the proliferation of human erythroid progenitors (erythroid burst-forming units IBFU-EI and colony-forming units [CFU-E]) in vitro. To examine the mechanism(s) underlying this inhibitory activity, the effect of different doses (50-10,000 U) of a highly purified preparation of recombinant DNA producfd human 'y-IFN on erythroid colony formation by normal human bone marrow BFU-E and CFU-E in the presence and absence of monocytes and/or T lymphocytes was studied. The addition of y-IFN to whole marrow caused suppression of BFU-E (6-65%) and CFU-E (31-79%) in a dose-dependent fashion. This inhibition occurred both with the direct addition of y-IFN to the culture plates as well as by the preincubation of marrow cells with y-IFN followed by the washing of the cells; at the highest concentration of y-IFN (10,000 U), near-maximal inhibition of colony formation occurred with as little as 15 min of preexposure (BFU-E, 50%; CFU-E, 81%). Removal of monocytes and/or T lymphocytes before the addition of "y-IFN significantly reduced the inhibitory effects of this lymphokine (BFU-E, -1 to 38%; CFU-E, -8 to 67%). Co-culture of purified autologous monocytes or T cells preexposed to 'y-IFN with monocyte and T cell-depleted marrow cells resulted in highly significant inhibition of erythroid colony formation even when these treated cells comprised <1% of the total nucleated cell populations in culture. The inhibitory action of y-IFN was not prevented or reversed by erythropoietin. These results demonstrate that the inhibitory effects of -y-IFN on erythropoiesis are mediated to a significant degree through accessory cell populations, and suggest that 'y-IFN may represent a useful tool in the study of the role of immunocompetent cells in the regulation of erythropoiesis in vitro.