A primary signaling cascade responsible for the expression of cytokine- stimulated immediate early genes involves the activation of the Jak/Stat pathway. In addition to being tyrosine-phosphorylated, several signal transducers and activators of transcription (Stats), including Stat1α, Stat3, and Stat4, are phosphorylated on a conserved serine residue, which is a consensus phosphorylation site for mitogen-activated protein kinases (MAPKs). Serine phosphorylation of Stat1α is required for maximal transcriptional activation of early response genes by interferon γ (IFNγ) as well as the antiviral and antigrowth actions of this cytokine. Incubation of cells with either IFNγ or oncostatin M (OSM) activates Raf-1, a serine/threonine kinase responsible for the ultimate activation of p42 MAPK. To examine whether any of the signaling components that are required for activation of the Jak/Stat pathway are also necessary for activation of Raf- 1 by IFNs and OSM, we examined activation of Raf-1 in cell lines that are deficient in either Stat1α or Stat2. Unexpectedly, incubation of Stat1- deficient, but not Stat2-deficient cells with IFNγ or OSM for 5 min displayed no increase in Raf-1 activity. In peripheral blood lymphocytes Raf- 1 was associated with Stat1, and this interaction was disrupted after incubation of cells with IFNγ. Stat1-negative cells reconstituted with either Stat1α or Stat1α with a point mutation in the site where it is serine-phosphorylated displayed normal activation of Raf-1 by IFNγ and OSM. However, activation of Raf-1 was not observed in lines that expressed Stat1α containing a mutation in its tyrosine phosphorylation site or in its SH2 domain. These results provide the first example of a novel role of Stat1α not as a transcription factor, but as a protein which may function to scaffold signaling components required for activation of the distinct Raf/MEK/MAPK signaling cascade.