Mycobacterium bovis infection produces tubercular lymphadenitis in the head lymphatics of cattle and deer, in addition to pulmonary disease. A low-dose intra-tonsilar infection model that establishes tuberculosis (Tb) lymphadenitis in cattle and deer is characterised in this study. Intra-tonsilar infection of red deer (500 cfus of M. bovis) was monitored longitudinally at 6-week intervals over a period of 23 weeks. Lesion characteristics, bacteriological and immunological parameters were assessed, and compared against those observed in cattle at 20 weeks post-infection, where the latter were infected with 500 or 5000 cfus of M. bovis. Intra-tonsilar inoculation of M. bovis established infection in >90% of deer and cattle, with lesion frequencies at the draining sentinel lymphatic site (left medial retropharyngeal node) of 68-86% and tissue bacterial burdens >3.5 logs/g of tissue, the tonsil being a major site of M. bovis persistence in deer only. Mineralisation occurred at lesion sites in both species in the later stages (18-23 weeks) of infection, with extensive coarse mineralisation observed mainly in cattle. The severity of infection or disease in cattle that received the higher or lower dose of M. bovis did not differ markedly. Pathogen-induced cellular immune response (lymphocyte transformation) and humoral responses (IgG and IgG(1) anti-mycobacterial antibodies) were recorded in both species, and the magnitude of these was noticeably amplified by skin tuberculin testing. IgG(1) antibodies were detectable within 6 weeks post-inoculation in deer and could be associated with early detection of lymphadenitis. Deer and cattle show similar levels of susceptibility to M. bovis infection.