The interferon-alpha (IFN-alpha) regulated mouse Ifi54/Ifi56 gene family, which is composed of at least four members (Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2), was isolated and characterized. In addition, the chromosomal localization of the four genes was determined. The Ifi54 and Ifi56 genes show an identical organization. Both are composed of a very small first exon and a second exon, which contains the complete open reading frame, except for the ATG start codon and the first two nucleotides of the second codon. In both genes, the two exons are separated by a small intron (5 and 2.5 kb, respectively). Expression of both genes is rapidly induced by IFN-alpha (within 2 h). The Ifi54 promoter region contains two sequences, which are closely related to the interferon stimulated response element (ISRE) consensus sequence (ISRE 1, GGTTTCAATTTCT, and ISRE 2, AGTGTTACTTTCT). The two elements are located directly adjacent to each other. A similar organization was recently established for the hamster Ifi54 promoter (Bluyssen et al., 1994). However, the mouse promoter is 70% less active than the hamster promoter. It turned out that ISRE 2 is hardly active, due to the G at position 4, which is a T in the hamster Ifi54 ISRE 2 and in the ISRE consensus sequence. The Ifi56 promoter region contains at a similar position two functional ISREs of identical strength (ISRE 1, AGTTTCAGTTTCT, and ISRE2, AGTTTCACTTTCC). In the Ifi56 promoter, the two ISRE motifs are separated by 6 bp. In addition to the Ifi56 gene, parts of two closely related genes (Ifi56-ps1 and Ifi56-ps2) were isolated. Both fragments contain an Ifi56-related open reading frame. However, we were unable to isolate the presumed first exon of Ifi56-ps1 and Ifi56-ps2, nor could we show expression of the genes. The Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2 genes could all be assigned to mouse chromosome 19D1, suggesting a tight clustering.