IL-15 is an immunostimulatory cytokine trans-presented with the IL-15 receptor alpha-chain to the shared IL-2/IL-15Rbeta and common gamma-chains displayed on the surface of T cells and NK cells. To further define the functionally important regions of this cytokine, activity and binding studies were conducted on human IL-15 muteins generated by site-directed mutagenesis. Amino acid substitutions of the asparagine residue at position 72, which is located at the end of helix C, were found to provide both partial agonist and superagonist activity, with various nonconservative substitutions providing enhanced activity. Particularly, the N72D substitution provided a 4-5-fold increase in biological activity of the IL-15 mutein compared with the native molecule based on proliferation assays with cells bearing human IL-15Rbeta and common gamma-chains. The IL-15N72D mutein exhibited superagonist activity through improved binding ability to the human IL-15Rbeta-chain. However, the enhanced potency of IL-15N72D was not observed with cells expressing the mouse IL-15Ralpha-IL-15Rbeta-gamma(c) complex, suggesting that this effect is specific to the human IL-15 receptor. The enhanced biological activity of IL-15N72D was associated with more intense phosphorylation of Jak1 and Stat5 and better anti-apoptotic activity compared with the wild-type IL-15. IL-15N72D superagonist activity was also preserved when linked to a single-chain TCR domain to generate a tumor-specific fusion protein. Thus, the human IL-15 superagonist muteins and fusions may create opportunities to construct more efficacious immunotherapeutic agents with clinical utility.