When interleukin-2 (IL-2) was added to immature, low-ploidy (greater than 80% 2N+4N) megakaryocytes generated in IL-3 and stem cell factor (SCF)-containing liquid cultures of blood mononuclear cells highly enriched in hematopoietic progenitors, a 2- to 6-fold increase in the absolute number of polyploid (more than 8N) megakaryocytes was noted. This effect was found to be indirect and was mediated through natural killer (NK) cells that constitute the major lymphoid cell contaminating day 6 megakaryocyte cell populations. IL-2 had no effect on megakaryocytes generated from CD34(+) cells stimulated with IL-3 and SCF. However, medium conditioned by IL-2-stimulated, but not resting, NK cells (NKCM) contained a trypsin-sensitive factor capable of increasing 2- to 5-fold the number of polyploid megakaryocytes generated in vitro from IL-3 and SCF-stimulated CD34(+) cells. The activity in NKCM was dose dependent and could not be neutralized by an excess of antibodies to IL-6, IL-11, leukemia inhibitory factor (LIF), gp130, stromal cell derived factor-1a (SDF-1a), and thrombopoietin (TPO). Addition of IL-11, but not TPO, to NKCM-containing cultures resulted in further augmentation of polyploidy, with the generation of 50% to 70% polyploid megakaryocytes with a modal ploidy of 16N. This factor is distinct from TPO because it induces endomitosis in IL-3-generated megakaryocytes in vitro, whereas TPO does not, and its activity on megakaryocyte ploidy is not altered by optimal concentrations of TPO. In addition, no message for TPO is detectable in IL-2-stimulated NK cells by reverse transcription-polymerase chain reaction. These findings indicate that IL-2-stimulated NK cells produce a novel peptide, distinct from TPO, IL-6, IL-11, LIF, other gp130-associated interleukins, and SDF1a, that can induce in vitro endomitosis in immature human megakaryocytes in the presence of IL-3 and SCF.