We report the molecular characterization of the porcine IFN-omega multigenic family which appears to comprise six to seven loci. Five of these loci were cloned and sequenced, revealing two pseudogenes (psi PoIFN-omega 1 and -omega 2) and three genes with an intact reading frame (PoIFN-omega 3, -omega 4, and -omega 5) that could encode preproteins of 179-190 amino acids including a putative signal peptide of 23 residues. By comparison of porcine IFN-omega coding sequences to those of known mammalian counterparts, it appeared that porcine sequences contain an in-frame five-codon deletion (between positions 113 and 117) in a region of relatively high sequence variability. In vitro transcription and translation of the three potentially functional reading frames gave rise to proteins with antiviral activity, showing that the porcine-specific deletion does not abolish the biological activity. Comparative analysis of flanking sequences revealed unique features of the 3' untranslated region of IFN-omega 4 gene: It contained a consensus estrogen-responsive element (ERE) in the vicinity of an extensive A-T-rich sequence known to serve a specific regulatory role in the expression of many genes involved in the inflammatory response. This finding suggests that the expression of the functional members of the porcine IFN-omega family may be mediated through different mechanisms. The expression of these genes was studied by Northern blot analysis of mRNAs from pig conceptus (days 14-20). Five bands of poly(A)+ RNAs hybridized with an IFN-omega probe provided the stringency was low, suggesting that a distinct family of IFN-omega-related genes were expressed by porcine trophoblastic cells during early gestation.