A scientific review of the recent sharp increase in bovine tuberculosis in Great Britain has concluded that the development of a cattle vaccine holds the best prospect for long-term disease control. It is important to develop a diagnostic test that differentiates between vaccinated and Mycobacterium bovis-infected animals, to ensure that test-and-slaughter control strategies can continue alongside vaccination. The mycobacterial antigens ESAT-6, MPB64, and MPB83 are expressed at high levels in M. bovis but are expressed at low levels or not at all in bacille Calmette-Guérin (BEG) Pasteur. Promiscuous bovine T cell epitopes of these antigens were identified and formulated into a peptide cocktail. This cocktail and a cocktail composed of recombinant forms of the 3 antigens was able to distinguish cattle infected with virulent M. bovis from those vaccinated with BeG and from those sensitized to avian tuberculin in lymphocyte transformation and interferon-γ assays.