Cytotoxic T-cell responses are thought to play a significant role in the host defence against mycobacterial infections. Little is understood about such responses of cattle to Mycobacterium bovis, the causative agent of bovine tuberculosis. The work described in this report demonstrates the activity of cytotoxic cells during experimental infection of cattle with M. bovis. The cytotoxic cells were found to have the ability to specifically lyse macrophages infected with M. bovis and were detected in peripheral blood lymphocytes after in vitro re-exposure to M. bovis. Cytotoxic activity was detected 4 weeks after experimental infection with M. bovis; a similar level of activity was maintained during the infection and it was mediated by both WC1+gammadelta and CD8+ T cells. In addition, inhibition of the growth of M. bovis within infected macrophages was detected when they were exposed to cultures containing M. bovis-specific cytotoxic cells. The ability to detect cytotoxic cells after infection of cattle with M. bovis will allow their activity to be measured during vaccination trials. Correlation of cytotoxic activity with disease outcome may aid in the design of new vaccines and vaccination strategies.