Human IFN-γ-inducible protein, 10 kDa (hIP-10) and murine IP-10 (mIP-10) genes are induced by IFN-γ alone, and synergistically induced by TNF-α and IFN-γ. Upstream regions of the human and murine genes contain conserved regulatory motifs, including an IFN-stimulated response element (ISRE), which governs response of the mIP-10 gene to IFN-γ. Trans-acting factors mediating the IFN-γ response via ISRE remain incompletely defined. We examined ISRE-binding factors in the regulation of the hIP-10 gene. The requirement of p48 for hIP-10 induction by IFN-γ, with or without TNF-α, was demonstrated using p48-deficient U2A cells. An hIP-10 promoter-reporter mutant (mISRE3) that was relatively deficient for binding a related factor, IFN regulatory factor-1 (IRF-1) but competent for binding p48, was induced as well as the wild-type hIP-10 promoter, supporting the interpretation that p48 played a necessary and sufficient role in hIP-10 transcription. Genomic in vivo footprinting revealed IFN-γ/TNF-α-inducible binding at the ISRE consistent with the presence of p48 and associated factors, but not with IRF-1. Induction of hIP-10 by TNF-α/IFN-γ also required NFκB binding sites, which were protected in vivo and bound p65 homodimeric NFκB in vitro. These results documented the essential role of p48 (complexed with STAT-1α) for induction and sustained transcription of the IP-10 gene, strongly suggesting that IRF-1 is not required for IP-10 induction by these inflammatory cytokines.