IL-15 is a pleiotropic cytokine that plays important roles in both innate and adaptive immunity. It is associated with a range of immunopathology, including rheumatoid arthritis and allograft rejection. IL-15 functions through the trimeric IL-15R complex, which consists of a high affinity binding α-chain and the common IL-2R β-and γ-chains. Characterization of IL-15/IL-15R interactions may facilitate the development of improved IL-15 antagonists for therapeutic interventions. We previously constructed soluble murine IL-15Rα (sIL-15Rα) by deleting the cytoplasmic and transmembrane domains. To localize the functional domain of IL-15Rα, we have now constructed various truncated versions of sIL-15Rα. The shortest region retaining IL-15 binding activity is a 65-aa sequence spanning the Sushi domain of IL-15Rα. Sushi domains, common motifs in protein-protein interactions, contain four cysteines forming two disulfide bonds in a 1-3 and 2-4 pattern. Amino acid substitution of the first or fourth cysteine in sIL-15Rα completely abolished its IL-15 binding activity. This also abrogated the ability of sIL-15Rα to neutralize IL-15-induced proinflammatory cytokine production and anti-apoptotic response in vitro. Furthermore, the mutant sIL-15Rα lost its ability to inhibit carrageenan-induced local inflammation and allogenic cell-induced T cell proliferation and cytokine production in vivo. Thus, the Sushi domain is critical for the functional activity of sIL-15Rα.