To identify the epitopes in human interleukin-15 (IL-15) that are responsible for binding to the interleukin-15 receptor α chain, antibody and receptor mapping by peptide scanning and site-directed mutagenesis was used. By using peptide scanning, we identified four regions in IL-15. The first region (85 CKECEELEEKN 95) is located in the C-D loop and is recognized by a set of non-inhibitory antibodies. The second region (102 SFVHIVQMFIN 112) is located in helix D and is recognized by two antibodies that are inhibitory of IL-15 bio-activity but not of IL-15 binding to IL-15Rα. The two remaining regions react with a recombinant soluble form of the IL-15Rα; the first (44 LLELQVISL 52 , peptide 1) corresponds to a sequence located in the B-helix and the second (64 ENLII 68 , peptide 2) to a sequence located in helix C. The latter is also contained in the epitope recognized by an antibody (monoclonal antibody B-E29) that prevents IL-15 binding to IL-15Rα. By site-directed mutagenesis, we confirmed that residues present in peptide 1 (Leu-45, Glu-46, Val-49, Ser-51, and Leu-52) and peptide 2 (Leu-66 and Ile-67) are involved in the binding of IL-15 to IL-15Rα. Furthermore, the results presented indicate that residues in the second peptide (Glu-64, Asn-65, and Ile-68) participate in IL-2Rβ recruitment. This finding could have implications for the dynamics of receptor assembly. These results also indicate that the modes of interaction of IL-15 and IL-2 with their respective α chains are not completely analogous. Finally, some of the IL-15 mutants generated in this study displayed agonist or antagonist properties and may be useful as therapeutic agents.