The aim of this work was to determine the minimum infective dose of Mycobacterium bovis necessary to stimulate specific immune responses and generate pathology in cattle. Four groups of calves (20 animals) were infected by the intratracheal route with 1,000, 100, 10, or 1 CFU of M. bovis. Specific immune responses (gamma interferon [IFN-γ] and interleukin-4 [IL-4] responses) to mycobacterial antigens were monitored throughout the study, and the responses to the tuberculin skin test were assessed at two times. Rigorous post mortem examinations were performed to determine the presence of pathology, and samples were taken for microbiological and histopathological confirmation of M. bovis infection. One-half of the animals infected with 1 CFU of M. bovis developed pulmonary pathology typical of bovine tuberculosis. No differences in the severity of pathology were observed for the different M. bovis doses. All animals that developed pathology were skin test positive and produced specific IFN-γ and IL-4 responses. No differences in the sizes of the skin test reactions, the times taken to achieve a positive IFN-γ result, or the levels of the IFN-γ and IL-4 responses were observed for the different M. bovis doses, suggesting that diagnostic assays (tuberculin skin test and IFN-γ test) can detect cattle soon after M. bovis infection regardless of the dose. This information should be useful in modeling the dynamics of bovine tuberculosis in cattle and in assessing the risk of transmission.