Bovine tuberculosis (bTB) is increasing in incidence in the UK. Effective control strategies could involve vaccination; BCG, either alone or in prime-boost strategies, remains the most effective vaccine against bovine tuberculosis. However, BCG vaccination of cattle would require development of diagnostic tests able to accurately discriminate Mycobacterium bovis-infected from BCG-vaccinated animals. Herein, we demonstrate that the detection of secreted IFN-γ following short term culture (4 h) of whole blood with purified protein derived from M. bovis (PPD-B) allows such discrimination. This reflects, in part, the differential kinetics of IFN-γ secretion in infected compared to vaccinated cattle. This is the first study to demonstrate that accurate, rapid distinction of BCG-vaccinated from M. bovis-infected cattle can be achieved in a short time period without the need for production of M. bovis-specific antigens, complex antigen mixtures or extensive laboratory procedures. We were also able to detect PPD-specific IFN-γ release during short term culture of blood from a number of humans with active TB indicating that this test may have wider application and is potentially useful for the rapid diagnosis of disease in humans.