We evaluated the usefulness of interferon- release assays (IGRAs) in the diagnosis of active tuberculosis (TB) in routine clinical practice. We retrospectively assessed the diagnostic usefulness of the QuantiFERON-TB Gold In-Tube (QFT-IT) test in 519 consecutive patients who were suspected of having active pulmonary TB and had QFT-IT test results. Active pulmonary TB was diagnosed in 242 patients (47%). The sensitivity in the diagnosis of active pulmonary TB was higher with the QFT-IT test (84%) than with the tuberculin skin test (TST; 74%; p=0.003). The negative predictive value in the diagnosis of active pulmonary TB was also higher with the QFT-IT test (79%) than with the TST (70%; p=0.023). Among 59 patients with non-tuberculous mycobacterial lung disease, the QFT-IT test was positive in 29 patients (49%). In conclusion, the high negative predictive value of the QFT-IT suggests a supplementary role for this test in the diagnostic exclusion of active TB. However, negative QFT-IT test results should not be used alone to exclude active TB. Additionally, the QFT-IT test has limited usefulness in differentiating active pulmonary TB from non-tuberculous mycobacterial lung disease in areas with a high prevalence of latent tuberculosis infection.