The immunosuppressive drug rapamycin suppresses T-cell activation by impairing the T-cell response to lymphokines such as interleukin-2 (IL- 2) and interleukin-4 (IL-4). In addition, rapamycin blocks the proliferative response of cell lines to a variety of hematopoietic growth factors, including interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- colony stimulating factor (GM-CSF), and kit ligand (KL), suggesting that it should be a strong inhibitor of hematopoiesis. In this report, we studied the effects of rapamycin on different hematopoietic cell populations in vitro and in vivo. In vitro, rapamycin inhibited the proliferation of primary bone marrow cells induced by IL-3, GM-CSF, KL, or a complex mixture of factors present in cell-conditioned media. Rapamycin also inhibited the multiplication of colony-forming cells in suspension cultures containing IL-3 plus interleukin-1 (IL-1) or interleukin-11 (IL-11) plus KL. In vivo, treatment for 10 to 28 days with high doses of rapamycin (50 mg/kg/d, orally) had no effect on myelopoiesis in normal mice, as measured by bone marrow cellularity, proliferative capacity, and number of colony-forming progenitors. In contrast, the same treatment strongly suppressed the hematopoietic recovery normally seen 10 days after an injection of 5-fluorouracil (5- FU; 150 mg/kg, intravenously [i.v.]). Thus, rapamycin may be detrimental in myelocompromised individuals. In addition, the results suggest that the rapamycin-sensitive cytokine-driven pathways are essential for hematopoietic recovery after myelodepression, but not for steady-state hematopoiesis.