As part of our ongoing program to expand immunological reagents available for research in cattle, we developed a monoclonal antibody (mAb) to bovine interleukin-17A (IL-17A), a multifunctional cytokine centrally involved in regulating innate and adaptive immune responses. Initial comparative studies demonstrated the mAb recognizes a conserved epitope expressed on orthologues of IL-17A in sheep, goats and pigs. Comparative flow cytometric analyses of lymphocyte subsets stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin revealed differences in expression of IL-17A by CD4, CD8, and γδ T cells across ruminants and swine species. Results in cattle showed the largest proportion of IL-17A+ cells were CD4+ followed by γδ and CD8+ T cells. Further analysis revealed the IL-17A+ γδ T cell subset was comprised of WC1.1+, WC1.2+, and WC1- subsets. Analysis of the IL-17A+ CD8+ T cell subset revealed it was comprised of αβ and γδ T cell subsets. Results in sheep and goats revealed IL-17A is expressed mainly by CD4+ and CD8+ T cells, with little expression by γδ T cells. Analysis of IL-17A+ CD8+ T cells showed the majority were CD8+ αβ in sheep, whereas they were CD8+ γδ in goats. The majority of the sheep and goat IL-17A+ γδ T cells were WC1+. Results obtained in swine showed expression of IL-17A by CD4, CD8, and γδ T cell subsets were similar to results reported in other studies. Comparison of expression of IL-17A with IFN-γ revealed subsets co-expressed IL-17A and IFN-γ in cattle, sheep, and goats. The new mAb expands opportunities for immunology research in ruminants and swine.