Tuberculosis (TB) remains a global health problem in humans and animals, and improved diagnostic methods are needed urgently. This study examined the potential of an interferon-gamma blood test based on a recently identified low-molecular-mass secreted protein antigen, ESAT-6, for early detection of bovine TB. It was found that field cases of bovine TB and experimentally infected cattle exhibited strong in vitro interferon-y responses directed toward this antigen. Of importance, ESAT-6 reactivity was found to discriminate between cattle infected with TB and cattle sensitized by environmental mycobacteria, and the gene encoding this molecule was demonstrated to be absent from >90% of the nontuberculous mycobacterial strains isolated from healthy sensitized cattle. These results demonstrate the feasibility of using single defined antigens for the highly specific diagnosis of TB.