Interleukin 4 (IL-4) is a potent, pleiotropic lymphokine that affects a variety of cells, especially those of hematopoietic origin. Although murine and human IL-4 are homologous proteins, they display a species specificity in which murine IL-4 acts only upon mouse cells, and human IL-4 only upon human cells. We have used a mutagenesis strategy to define both the structural determinants of this specificity and a receptor binding domain of murine IL-4. To do this, we developed convenient solid-phase binding assays for mouse and for human IL-4, each utilizing receptor-immunoglobulin fusion proteins and alkaline phosphatase-tagged ligands. These were employed to assess the receptor binding activities of wild type and mutant forms of IL-4. In a separate biological assay, we measured the ability of each version of IL-4 to induce proliferation of a cultured mouse T-cell line. By replacing regions of mouse IL-4 with homologous segments of human IL-4, we found that the amino-terminal 16 residues and the carboxyl-terminal 20 residues of murine IL-4 are required for species-specific receptor binding as well as for T-cell proliferation. A major portion of the amino acid sequence between these regions can be substituted between mouse and human without loss of receptor binding or biological activity. Further, alanine-scanning mutagenesis revealed specific residues in the amino- and carboxyl-terminal regions (Glu-12, Ile-14, Leu-104, Asp-106, Phe-107, and Leu-111) that bear side chains critical for function. An analysis of the carboxyl-terminal region of murine IL-4 and its comparison with carboxyl-terminal regions of other related cytokines suggest an evolutionary conservation of structural and functional features.