Technical Abstract: The purpose was to evaluate antigen-specific and polyclonal proliferative and activation-associated responses by blood mononuclear cells from Mycobacterium bovis-infected reindeer. Peripheral blood mononuclear cells (PBMC) from M. bovis-infected (n = 10) and non-infected reindeer (n = 4) were stimulated with a recombinant early secretory antigenic target-6 and culture filtrate protein-10 fusion protein (ESAT6:CFP10, antigens specific to tuberculous mycobacteria), M. bovis purified protein derivative, pokeweed mitogen, or medium alone and evaluated for proliferation and activation marker expression by flow cytometry. gd TCR**+ and CD8**+ cells, but not CD4**+ cells, from M. bovis-infected reindeer proliferated in response to specific antigen stimulation. Expression (i.e., mean fluorescence intensity) of CD44 was increased and CD62L decreased on proliferative as compared to non-proliferative PBMC in antigen- and mitogen-stimulated cultures. In response to stimulation with rESAT6:CFP10, MHC II fluorescence intensity was increased on CD4**+,'gd TCR**+, CD172a**+, and IgM**+ cells as compared to non-stimulated cells from infected, but not non-infected, reindeer. rESAT6:CFP10 stimulation also induced expansion of a CD172a**+,MHC II**+ population within PBMC cultures from M. bovis-infected reindeer. Despite a moderate challenge dose and an extended duration of infection, experimental infection of reindeer was generally limited to lymph nodes draining the inoculation site. Therefore, in vitro findings presented in this study may be predictive of host resistance to tuberculous mycobacteria.