Interleukin 12 (IL-12), or natural killer cell stimulatory factor (NKSF), has multiple biologic effects on T lymphocytes and natural killer cells. In this study, we evaluated the effect of IL-12 on human hematopoiesis by analyzing the growth of CD34+33- cells in methylcellulose culture with or without serum. Blood cells were collected by apheresis from an expanded stem cell pool during the rebound phase of hematopoiesis after chemotherapy and purified by combined purification procedures to yield target CD34+33- cells. IL-12, by itself or in combination with IL-3 and erythropoietin (Epo), had no effect on the formation of colonies from the unfractionated blood mononuclear cells. In the presence of fetal bovine serum (FBS), IL-12 increased the number of colony forming units for granulocyte-macrophage (CFU-GM) from CD34+33- cells which responded to IL-3, but not the number of CFU-GM from those which responded to stem cell factor (SCF) or granulocyte colony stimulating factor (G-CSF). This effect was not abolished by the addition of monoclonal antibodies against tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-1 alpha, IL-6, G-CSF, or granulocyte/macrophage CSF (GM-CSF). However, the stimulatory effect of IL-12 was abolished in secondary plating in serum-containing methylcellulose culture after short-term (48 h) liquid incubation with IL-3.(ABSTRACT TRUNCATED AT 250 WORDS)