Interleukin 2 (IL-2) mediated signalling results from ligand binding and subsequent hetero-dimerization of IL-2rβ and γc. The high-affinity IL-2 receptor (IL-2r) is a heterotrimer comprised of the IL-2rα, IL-2rβ and γc subunits. Whereas human IL-2 effectively binds to either human or murine lymphocytes, murine IL-2 binds with markedly higher affinity to murine receptor complexes than to human complexes. Using cell lines stably expressing heterotrimeric IL-2r that vary in the species origin of individual subunits, we have demonstrated that IL-2rα is primarily responsible for the species specificity of IL-2 binding. Studies of ligand binding to the low affinity receptor demonstrated that IL-2rα displays a similar species preference to the heterotrimeric complex. Moreover, differences in ligand binding are reflected in differences in proliferation. A cell line expressing human IL-2rα and IL-2rβ along with murine γc vigorously proliferated only in response to human IL-2 at low doses, while both human and murine IL-2 stimulated proliferation of a cell line containing murine IL-2rα (as well as human IL-2rβ and murine γc). Therefore, IL-2rα is the chain primarily responsible for the species specificity of ligand binding.