A novel, whole blood interferon-gamma (IFN-gamma) assay was evaluated to determine its suitability for detecting Mycobacterium tuberculosis exposure in intravenous drug users with or without human immunodeficiency virus (HIV) infection. Whole heparinized blood was incubated overnight in separate wells with tuberculin purified protein derivative (PPD), saline, and mitogen controls. Levels of IFN-gamma in plasma supernatants were determined by rapid ELISA. Participants were then administered the tuberculin skin test (TST) and tested for cutaneous anergy. The whole blood IFN-gamma test agreed (89%-100%) with a positive TST in both HIV-seropositive and -seronegative subjects, but reactivity to PPD was more detectable by the whole blood assay among those with negative TSTs or anergy. TST induration diameter and IFN-gamma responses were correlated (Spearman's p = .45, P = .0001), but both responses were blunted by HIV infection. In summary, tuberculin reactivity appears to be more detectable by the whole blood IFN-gamma assay than by TST, and the assay requires no return visit for test reading.