The p69/71 2-5A synthetase is an interferon-inducible enzyme that polymerizes ATP to form 2'-5'-linked oligoadenylates when activated by double-stranded RNA, A genomic clone was isolated that contained 12.5 kb of the 5'-flanking region and the first exon of the p69/71 2-5A synthetase gene, The major transcriptional start site was mapped to an A residue located 84 bp upstream of the translational start site within a sequence that matches both a consensus ISRE and an Inr element. Sequencing of the region 972 bp upstream of the translation start site revealed 4 imperfect direct repeats of 70 to 80 bp that contain several putative regulatory elements, This region does not have a TATA or CAAT box but does contain two IRF-1-like elements, an Ets-1 motif, an AP-1 site, an Spl binding site, an NF-kappa B-binding site, and a palindrome containing two overlapping NF-IL-6 consensus motifs in opposite orientation, The region -480 to -850 bp contains PuF, UBP-1, and PEA 3 motifs and another NF-IL-6 motif adjacent to an E2A-binding site, The 5'-flanking sequence binds proteins in DNase I foot printing assays and is a functional interferon-inducible promoter that requires multiple elements for maximal constitutive and interferon-inducible expression.