The effects of recombinant human macrophage colony-stimulating factor (rhCSF-1) in long-term marrow cultures (LTMC) established from normal bone marrow cells were examined. When added during the first 3 weeks of culture (every second day, at 15 ng/mL), rhCSF-1 strongly inhibited the growth of all hematopoietic progenitors analyzed (colony-forming unit- MIX [CFU-MIX], CFU-granulocyte macrophage [CFU-GM], CFU-M, CFU-G, burst- forming unit-erythroid). Paralleling the inhibition of progenitors was the complete loss of adipocytes from the stromal layer of rhCSF-1- treated cultures. The inhibitory effect of rhCSF-1 correlated in all instances with the accumulation in the supernatants of these cultures of an activity (different from CSF-1) that inhibited colony formation in semisolid cultures. When addition of rhCSF-1 was delayed 3 weeks, its inhibitory effects were significantly reduced, which correlated with reduced inhibitory activity detected in the supernatants. Analysis of CSF-1 concentration by radioreceptor assay confirmed that added rhCSF-1 increased culture CSF-1 levels and showed that the decreased inhibition observed when rhCSF-1 is added later in culture was not due to decreased CSF-1 levels at that point. In contrast, the ability of rhCSF-1 to inhibit hematopoiesis and accumulate inhibitory activity in LTMC correlated with its rate of utilization, much higher in the first 2 weeks of culture, when the stromal layer was being established, than later. These observations document the inhibitory effect of rhCSF-1 on all aspects of hematopoiesis conducted in cultures that simulate the hematopoietic microenvironment, demonstrate the importance of accessory/stromal cells in mediating the effects of rhCSF-1 in LTMC, and point to an inhibitory activity as the mediating agent.