The role of interferon-γ release assays in the diagnosis of active tuberculosis disease is uncertain, and recent guidelines do not support their routine use. We reviewed the clinical records of 415 patients who had a QuantiFERON-TB Gold In-Tube assay between 29 June 2005 and 28 October 2010 to determine its performance in the diagnosis of active tuberculosis disease in a low prevalence setting, specifically in human immunodeficiency virus (HIV) -positive and HIV-negative patients, those of UK and non-UK origin, and those with pulmonary and extrapulmonary disease. For the diagnosis of active tuberculosis disease the overall sensitivity of QuantiFERON-TB Gold In-Tube assay was 71.4% (95% CI 59.3–81.1), specificity was 81.0% (95% CI 75.5–85.6) and negative predictive value was 92.6% (95% CI 88.2–95.5). No significant difference in sensitivity was seen in culture-positive and culture-negative tuberculosis, in pulmonary and extrapulmonary disease, or with HIV infection. Specificity and negative predictive value were significantly higher in patients of UK origin compared with those of non-UK origin (89.3% (95% CI 83.3–93.3) and 97.1% (95% CI 92.7–98.9) versus 66.3% (95% CI 55.6–75.5) and 83.3% (95% CI 72.6–90.4)). Our study suggests that there may be a role for interferon-γ release assays in excluding active tuberculosis disease, particularly extrapulmonary disease, in patients originating from areas of low tuberculosis incidence, with a negative test highly predictive of a lack of active tuberculosis disease in this group. We cannot support the use of these assays in the diagnosis of active tuberculosis infection in patients from areas of higher incidence.