Interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) cooperate to induce the expression of many gene products during inflammation. The present report demonstrates that a portion of this cooperativity is mediated by synergism between two distinct transcription factors: signal transducer and activator of transcription 1 (STAT1) and nuclear factor κB (NF-κB). IFNγ and TNFα synergistically induce expression of mRNAs encoding interferon regulatory factor-1 (IRF-1), intercellular adhesion molecule-1, Mig (monokine induced by γ-interferon), and RANTES (regulated on activation normal T cell expressed and secreted) in normal but not STAT1-deficient mouse fibroblasts, indicating a requirement for STAT1. Transient transfection assays in fibroblasts using site-directed mutants of a 1.3-kilobase pair sequence of the IRF-1 gene promoter revealed that the synergy was dependent upon two sequence elements; a STAT binding element and a κB motif. Artificial constructs containing a single copy of both a STAT binding element and a κB motif linked to the herpes virus thymidine kinase promoter were able to mediate synergistic response to IFNγ and TNFα; such response varied with both the relative spacing and the specific sequence of the regions between these two sites. Cooperatively responsive sequence constructs bound both STAT1α and NF-κB in nuclear extracts prepared from IFNγ- and/or TNFα- stimulated fibroblasts, although binding of individual factors was not cooperative. Thus, the frequently observed synergy between IFNγ and TNFα in promoting inflammatory response depends in part upon cooperation between STAT1α and NF-κB, which is most likely mediated by their independent interaction with one or more components of the basal transcription complex.