Natural killer (NK) cells have a key role in the innate immune response against pathogens because of their cytotoxic properties and production of interferon-gamma (IFN-gamma). Some insight into ruminant NK cell biology has been gained through the characterization of bovine NK cells as NKp46(+)/CD3(-) cells. However, ovine NK cells have been little studied because of the lack of specific antibodies. Most NK cells in humans and cattle express CD16. We found that an antibody against human CD16 that cross-reacts with bovine NK cells also recognizes cell populations in ovine peripheral blood mononuclear cells. Using double labelling with CD14 revealed the same profile as described in other species, and we identified a putative NK cell population. We therefore sorted this ovine CD16(+)/CD14(-) cell population and tested it for NK cell characteristics. More than 80% of sorted CD16(+)/CD14(-) cells expressed perforin. After a week of culture in the presence of IL-2 and IL-15, ovine CD16(+)/CD14(-) cells had become large cells with intra-cytoplasmic granules containing perforin, and the vast majority displayed an activated CD2(-/low)/CD25(+)/CD8(+) phenotype, as observed for bovine NKp46(+)/CD3(-) cells. Moreover, these cells expressed transcripts for the NKp46 receptor, and were cytotoxic in a CD16-mediated redirected lysis assay against a murine cell line, P815, and in a direct lysis assay against the ovine cell line, IDO5. Finally, ovine CD16(+)/CD14(-) cells having expanded for 7 days in culture secreted IFN-gamma in response to IL-12 in a dosedependent manner. Taken together, these findings led us to conclude that the ovine CD16(+)/CD14(-) lymphocyte sub-population displays the phenotype and functional characteristics of NK cells.