Interleukin-12 (IL-12) or natural killer cell stimulatory factor (NKSF), has multiple effects on T lymphocytes and natural killer cells. In this study, the effect of IL-12 on human hematopoiesis was studied by analyzing the growth of CD34+ peripheral blood stem cells (PBSC), in steady state. In the presence of Epo, IL-12 alone or in combination with IL-3 or SCF had no effect on the formation of colonies from CD34+ cells. In culture with Epo, G-CSF, and IL-3, the effect of Flt3-ligand (FL) on CD34+ PBSC was investigated in the presence or absence of IL-12. No additional effect of IL-12 was observed when combined with FL. We evaluated 5-FU-treated human CD34+ PBSC proliferation in cultures with Epo, G-CSF, and IL-3, in the presence or absence of IL-12. No cytokine combination enhanced colony formation from 5-FU-treated CD34+ cells. However, in cultures of 5-FU-treated human CD34+ cells, the most efficient combination was IL-3 + Epo + G-CSF + Accessory cells (CD34-). Furthermore, IL-12 enhanced this colony formation significantly. To investigate whether immature CD34+ cells were responsible for FL or SCF, 5-FU-treated human CD34+ cells were cultured with or without IL-12. Whereas no synergistic effect was observed in combination with IL-12, SCF alone significantly enhanced colony formation. However, the colony number was found to be smaller than with the potent combination of accessory cells in the presence of IL-12. These results indicate that accessory cells, lost in CD34+ cell purification, could be partly responsible for an IL-12 effect on immature human PBSC proliferation.