Signaling by a wide variety of cytokines, including interferons, interleukins, and growth factors, involves activation of JAK kinases and Stat (Signal transducers and activators of transcription) proteins. At present, not much is known about the molecular mechanisms by which interleukin-5 (IL-5) exerts its diverse biologic effects. Human eosinophils are one of the most important target cells for IL-5 and were used here to study IL-5 signaling in a primary human cell. IL-5 induced rapid and transient tyrosine phosphorylation of JAK2. Moreover, IL-5 induced at least two DNA-binding complexes, using nuclear extracts from normal human eosinophils and the IL-6/interferon-gamma response element of the ICAM-1 promoter (ICAM-1 pIRE) in an electromobility shift assay. From supershift experiments it was concluded that one DNA- binding complex contained Stat1 alpha, probably as a homodimer. Both DNA-binding complexes were inhibited by a phosphotyrosine antibody (4G10), suggesting that tyrosine phosphorylation is required for complex formation. IL-3 and granulocyte-macrophage colony-stimulating factor induced, similar to IL-5, two DNA-binding complexes in human eosinophils, including Stat1 alpha. These data show for the first time that molecular mechanisms of IL-5 signaling in human eosinophils involve members of the JAK kinase family as well as members of the Stat family.