The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to the strategic development of new vaccines for the prevention of infectious diseases of livestock. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively poor in comparison. To address this gap in capability we have cloned bovine and ovine IL-17A and expressed the recombinant proteins in Chinese Hamster Ovary (CHO) cells. The recombinant IL-17A was shown to be functionally-active and reciprocally cross-reactive between cattle and sheep. On that basis we used the transfected CHO cells to screen a panel of commercially-available antibodies for ability to detect intracellular IL-17A expression and secretion. We demonstrate that an ELISA for bovine IL-17A also detects recombinant and native ovine IL-17A. Furthermore, the constituent polyclonal antibodies (pabs) of that ELISA can be used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that specifically discriminate between untransfected CHO cells and the IL-17A-transfected CHO cells by flow cytometry, whereas the capture pab was non-discriminatory. We used one of these mabs to detect intracellular IL-17A expression by activated PBMC from both cattle and sheep. By combining this mab with cell-surface phenotyping mabs we show that all of the major peripheral T cell subsets (CD4+ve, CD8+ve and WC1+ve[γδ]) in cattle and sheep are capable of expressing IL-17A. We also show that the proportions of cells expressing IL-17A within these T cell subsets are lower than those expressing IFN-γ under these stimulatory conditions. These data, therefore, provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.