Recognition of viral nucleic acids by vertebrate host cells results in the synthesis and secretion of type I interferons (IFN-alpha/beta), which induce an antiviral state in neighboring cells. We have cloned the genes and promoters of two type I IFNs from Atlantic salmon. Both genes have the potential to encode IFN transcripts with either a short or a long 5'-untranslated region, apparently controlled by two distinct promoter regions, PR-I and PR-II, respectively. PR-I is located within 116 nucleotides upstream of the short transcript and contains a TATA-box, two interferon regulatory factor (IRF)-binding motifs, and a putative nuclear factor kappa B (NFkappaB)-binding motif. PR-II is located 469-677 nucleotides upstream of the short transcript and contains three or four IRF-binding motifs and a putative ATF-2/c-Jun element. Complete and truncated versions of the promoters were cloned in front of a luciferase reporter gene and analyzed for promoter activity in salmonid cells. Constructs containing PR-I were highly induced after treatment with the dsRNA poly(I:C), and promoter activity appeared to be dependent on NFkappaB. In contrast, constructs containing exclusively PR-II showed poor poly(I:C)-inducible activity. PR-I is thus the main control region for IFN-alpha/beta synthesis in salmon. Two pathogenic RNA viruses, infectious pancreatic necrosis virus and infectious salmon anemia virus, were tested for their ability to stimulate the minimal PR-I, but only the latter was able to induce promoter activity. The established IFN promoter-luciferase assay will be useful in studies of host-virus interactions in Atlantic salmon, as many viruses are known to encode proteins that prevent IFN synthesis by inhibition of promoter activation.