We have utilized in vitro marrow culture techniques to evaluate the cytotoxicity for granulocytic progenitor cells of two highly purified human leukocyte interferon preparations. Concentration- and time-related decrements in granulocytic colony-forming capacity in agar occurred with human and mouse marrow. Although mouse marrow cells were less sensitive than were human cells, these data indicate lack of strict species specificity for the cell growth-inhibitory effects of interferon. Similar cytotoxicity was noted for normal and leukemic human clonogenic cells exposed to interferon for prolonged periods. The decrease in the proportion of granulocytic progenitor cells in DNA synthesis, which occurred at high concentrations, and the diminution by interferon of the cytotoxicity caused by cytosine arabinoside demonstrate that interferon decreases DNA synthesis of granulocytic progenitor cells. The lack of enhanced cytotoxicity for rapidly proliferating mouse post-endotoxin marrow cells indicates that interferon is not a cell cycle-stage-specific drug. These data seem useful for evaluating the suppressive effects of interferon on granulopoiesis and for devising clinical trials with this agent.