Gene transcription studies have identified dual roles for the cytokines IL-17A and IL-22 in bovine tuberculosis, where they show potential as both predictors of vaccine success and correlates of infection. To allow for a detailed investigation of the cell populations responsible for production of these cytokines, we have utilised a novel bovine IL-22 specific recombinant antibody for flow cytometry. Bovine tuberculin (PPDB) induced greater IL-22 and IL-17A production in Mycobacterium bovis (M. bovis)-infected cattle compared to non-infected controls, while PWM-induced cytokine levels were similar between the two groups. In M. bovis-infected animals, PPDB specific IL-22 and IL-17A responses were observed in both CD4+ T cell and πσT cell populations. Although both cytokines were detected in both cell types, IL-22/IL-17A double producers were rare and confined mainly to the πσT cell population. These results support previous gene transcription studies and extend the observation of increased IL-22 and IL-17A responses in M. bovis-infected animals to the level of protein production. We were also able to characterise the cell populations responsible for these disease-related cytokine responses. The data generated can be used to further our understanding of the immunopathology of bovine tuberculosis and to produce more sensitive and specific immune-diagnostic reagents.