TNFalpha levels are elevated in the marrows of patients with myelodysplastic syndrome (MDS) and are associated with high rates of apoptosis, which contributes to hematopoietic failure. We observed that exposure of human marrow stroma cell lines HS5 and HS27a to TNFalpha increases levels of IL-32 mRNA. IL-32, in turn, induces TNFalpha. Marrow stroma from patients with MDS expressed 14- to 17-fold higher levels of IL-32 mRNA than healthy controls. In contrast, cells from patients with chronic myelomonocytic leukemia (CMML) expressed only one tenth the level of IL-32 measured in healthy controls. Human KG1a leukemia cells underwent apoptosis when cocultured with HS5 stromal cells, but knockdown of IL-32 in the stromal cells by using siRNA abrogated apoptosis in the leukemia cells. IL-32 knockdown cells also showed dysregulation of VEGF and other cytokines. Furthermore, CD56(+) natural killer cells from patients with MDS and CMML expressed IL-32 at lower levels than controls and exhibited reduced cytotoxic activity, which was unaffected by IL-2 treatment. We propose that IL-32 is a marrow stromal marker that distinguishes patients with MDS and CMML. Furthermore, IL-32 appears to contribute to the pathophysiology of MDS and may be a therapeutic target.