A mouse genomic segment containing three new members of the murine interferon alpha (MuIFN-alpha) gene family was isolated from a fibroblastic cosmid library. A 4 kb EcoRI fragment contained a new MuIFN-alpha gene named MuIFN-alpha 8. The nucleotide sequence of the coding and flanking regions of this gene showed a high level of homology to those of known members of the MuIFN-alpha family. Transient expression of the MuIFN-alpha 8 gene in COS cells and oocyte translation of in vitro transcripts both led to a biologically active protein. The antiviral activity was neutralized by monoclonal and polyclonal MuIFN-alpha antibodies. Although the 5' flanking sequence shows features characteristic of an IFN regulatory region, the MuIFN-alpha 8 gene is not expressed in murine fibroblasts treated with Newcastle disease virus or poly(I).poly(C).