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 less than 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 cDNA corresponding to the three genes share 120 nucleotides of identical sequence and show greater than 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.