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Multiple species of type I interferon (IFN) were recently identified in catfish (CF) (Ictalurus punctatus). Herein we extend these studies and report the existence of two distinct type II IFN genes in channel CF. As with zebrafish and the green spotted pufferfish, the two CF IFN-gamma genes are dissimilar in sequence but closely linked on the same chromosome. One of the genes (IFN-gamma2) encodes two distinct messages that likely arose via alternative splicing at two closely spaced splice donor sites within the first intron. Sequence analysis indicates that CF IFN-gamma genes contain the hallmarks of authentic IFN-gamma including: (1) a conserved nuclear localization site at the C terminus (CF IFN-gamma2 only), (2) an IFN-gamma signature sequence, (3) six putative helical regions within the mature protein, (4) one or more potential glycosylation sites, and (5) multiple mRNA instability motifs within the 3' untranslated region. Moreover, well-characterized CF T and NK cell clones were shown to synthesize IFN-gamma transcripts. This is the first unequivocal demonstration in any lower vertebrate species that NK and T cells synthesize IFN-gamma and is consistent with results in mammalian systems where T cells and NK cells are the major sources of type II IFN production. Collectively, these studies indicate that Siluriformes possess two evolutionarily conserved IFN-gamma genes and demonstrate that CF possess three key elements of the innate immune response: NK cells and types I and II IFN.