Signal transducer and activator of transcription 6 (Stat6) and NF-κB are widely distributed transcription factors which are induced by different stimuli and bind to distinct DNA sequence motifs. Interleukin-4 (IL-4), which activates Stat6, synergizes with activators of NF-κB to induce IL-4-responsive genes, but the molecular mechanism of this synergy is poorly understood. Using glutathioneS-transferase pulldown assays and coimmunoprecipitation techniques, we find that NF-κB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. An IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4 when Stat6 and NF-κB proteins are coexpressed in human embryonic kidney 293 (HEK 293) cells. The same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-κB proteins in IL-4-stimulated I. 29μ B lymphoma cells. Furthermore, Stat6 and NF-κB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-κB. We conclude that the direct interaction between Stat6 and NF-κB may provide a basis for synergistic activation of transcription by IL-4 and activators of NF-κB.