ICSBP, a member of the interferon regulatory factor family, is expressed predominantly in lymphoid tissues and is induced by γ interferon (IFN-γ). We have studied the genomic organization of the murine ICSBP gene and its 5'upstream region. The murine ICSBP gene (Icsbp) is present as a single copy on chromosome 8 and consists of nine exons. Transcription initiates at two juxtaposed sites downstream from the TATA and CAAT boxes and produces two species of ICSBP mRNA (3.0 and 1.7 kb), presumably by differential usage of poly (A)+ signals. A sequence from-175 to-155 was identified to be an IFN response region that conferred IFN-γ induction upon a heterologous promoter in lymphoid cell line EL4. This region includes a motif, TTCNNGGAA, designated the palindromic IFN response element (pIRE), to which an IFN-γ-inducible, cycloheximide-sensitive factor (s) binds. A similar palindromic motif was found in the upstream region of the murine IRF-1 gene, the IFN-γ activation site of the guanylate-binding protein gene and the IFN-γ-responsive region of the Fc receptor type I gene, all of which competed with the pIRE for factor binding in gel mobility shift assays. We show that the pIRE binding factor reacts with the antibody against the 91-kDa subunit of ISGF3 α recently shown to bind to the IFN-γ activation site. These results suggest that this factor is related to the IFN-γ activation factor and contains the 91-kDa subunit of ISGF3 α. Taken together, pIRE represents an IRE that is distinct from the classical IFN-stimulated response element and that is capable of conferring IFN-γ induction through the binding of the 91-kDa ISGF3α subunit (or an antigenically similar molecule).