To create experimental systems that facilitate studies aimed at the responsiveness of hematopoietic progenitors to interleukin (IL)-6 in combination with IL-3, we introduced the human IL-6 receptor (hIL-6R) into the IL-3-dependent cell line 32D. For this purpose, a retroviral vector containing the hIL-6R cDNA was constructed. 32D parental cells did not respond to IL-6, neither alone nor in combination with increasing concentrations of IL-3, and did not express detectable numbers of IL-6R as determined by 125I-IL-6 binding. 32D/hIL-6R cells expressed high-affinity IL-6 binding and responded synergistically to IL-6 in combination with suboptimal amounts of IL-3 in DNA synthesis assays. In addition, IL-6 promoted the short-term survival of IL-3- responsive clonogenic 32D/hIL-6R cells. On the other hand, although introduction of hIL-6R resulted in the formation of high-affinity IL-6 receptor structures in the IL-2-dependent thymocyte cell line CTLL, CTLL/hIL-6R cells did not respond to IL-6 in synergy with IL-2. We conclude that 32D cells possess the intracellular machinery permissive for IL-6 signal transduction. Murine IL-3-dependent cell lines with ectopic IL-6 receptors can serve as models for dissecting the molecular basis of IL-6 responses in primitive hematopoietic cells.