Three functional members of the 1–8 gene family have been isolated on a single human genomic DNA fragment of less than 18 kb. The 1–8U and 1–8D genes are extremely similar: each is contained within a <2-kb fragment, has in its 5′flanking region two adjacent 14-base-pair sequences showing high similarity to interferon-stimulable response elements (ISREs) and has two highly related exons. The third gene (9–27) has a similar overall structure, shows substantial similarity to the 1–8s but has only one ISRE which is 3′ of two CCAAT boxes not present in the 1–8U and D genes. The cDNAs corresponding to the three genes share 120 nucleotides of identical sequence and show > 90% identity over 70% of the coding sequence. For the 1–8U and D genes the high similarity extends into the 5′ non-coding and flanking regions. The open reading frames encode polypeptides that are likely to be of very similar structure. Antiserum to a conserved peptide detects a polypeptide(s) of about 14 kDa on PAGE which separates into three components on isoelectric focussing. The 9–27 and 1–8U genes are highly interferon-inducible, the 1–8D gene is much less so. These differences are mimicked by the activities of the corresponding ISREs placed 5′ of a marker gene in expression constructs. They presumably reflect differences in the interaction of the ISREs with the various interferon-inducible and constitutive factors that govern the interferon response.