In this work, we report cDNA cloning of two type I interferons (IFNs) from the head kidney of Atlantic salmon, called SasaIFN-alpha1 (829 bp) and SasaIFN-alpha2 (1290 bp). Both translate into 175 amino acid precursor molecules showing 95% amino acid sequence identity. The precursors have a putative 23 amino acid signal peptide, which suggests that the mature Atlantic salmon IFNs contain 152 amino acids (18.2 kDa). Salmon IFN appears to have five alpha-helices, similar to mammalian and avian type I IFNs, and showed 45% sequence identity with zebrafish IFN, up to 29% identity with mammalian IFN-alpha sequences, and 17%-18% sequence identity with mammalian IFN-beta and chicken type I IFNs. Human embryonic kidney 293 cells transfected with the SasaIFN-alpha1 cDNA gene produced high titers of acid-stable antiviral activity, which protected salmonid cells against infectious pancreatic necrosis virus (IPNV) and also induced Mx protein in the cells. Poly(I)-poly(C) induced two IFN transcripts in head kidney of Atlantic salmon. Genomic IFN sequences contained four introns and five exons, which is different from the intronless type I IFN genes of birds and mammals.