Bovine tuberculosis is a major economic problem and a potential public health risk. Improved diagnostics like the gamma interferon (IFN-gamma) test with ESAT6 and/or CFP10 could contribute to the control program. We assessed IFN-gamma responses in zebu (Ethiopian Arsi breed) and Holstein cattle kept indoors or in a pasture to tuberculin purified protein derivative (PPD) and an ESAT6-CFP10 protein cocktail. Furthermore, the intensity and distribution of pathology of bovine tuberculosis were compared between the two breeds. Our data demonstrated significantly (all P < 0.02) higher IFN-gamma responses to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail in Holstein than in zebu cattle, while lesion severities in infected animals and tuberculin skin test responses did not differ significantly (P > 0.05) between the two breeds. Holstein cattle that were kept indoors produced significantly (all P < 0.01) higher IFN-gamma levels in response to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail than did Holstein cattle kept in a pasture. Moreover, lesion severity was significantly higher in Holstein cattle kept indoors (P = 0.001) than in those kept in the pasture. Lesions were localized predominantly in the digestive tract in cattle kept in a pasture, while they were localized in the respiratory tract in cattle kept indoors. In conclusion, in Holstein cattle, husbandry was a dominant factor influencing the severity of tuberculosis lesions and IFN-gamma responses to mycobacterial antigens compared to breed. A difference in the cellular immune response between zebu and Holstein cattle was observed, while tuberculosis lesion severities were identical in the two breeds, when both were kept in a pasture.