Using a serum-free culture system, we studied the interaction of interleukin 3 (IL-3) and interleukin 6 (IL-6) in the development of primitive hemopoietic progenitors. A time-course study showed that total colony formation supported by 200 U/ml of IL-3 alone failed to reach the level obtained by the combination of 40 ng/ml of IL-6 and IL-3 in culture containing bone marrow cells of 5-fluorouracil (5-FU) -treated mice. Extremely high concentrations (1,000 U/ml, 10,000 U/ml) of IL-3 also required the presence of IL-6 for the sufficient development of primitive progenitors. The depletion of phagocytic and T cells from crude bone marrow cells of 5-FU-treated mice did not influence the requirement for both factors. These results suggest the existence of primitive progenitors which require both IL-3 and IL-6 for development. The delayed addition of IL-6 to a culture initiated with IL-3 failed to restore total colony growth to the levels obtained by culture initiated with the two factors simultaneously. The results suggest that some primitive hemopoietic progenitors requiring both IL-3 and IL-6 for the substantial growth may be unable to survive in the presence of IL-3 alone.