ABSTRACT: BACKGROUND: False negative and indeterminate Interferon Gamma Release Assay (IGRA) results are a well documented problem. Cigarette smoking is known to increase the risk of tuberculosis (TB) and to impair Interferon-gamma (IFN-gamma) responses to antigenic challenge, but the impact of smoking on IGRA performance is not known. The aim of this study was to evaluate the effect of smoking on IGRA performance in TB patients in a low and high TB prevalence setting respectively. METHODS: Patients with confirmed TB from Denmark (DK, n = 34; 20 smokers) and Tanzania (TZ, n = 172; 23 smokers) were tested with the QuantiFERON-TB Gold In tube (QFT). Median IFN-gamma level in smokers and non smokers were compared and smoking was analysed as a risk factor for false negative and indeterminate QFT results. RESULTS: Smokers from both DK and TZ had lower IFN-gamma antigen responses (median 0.9 vs. 4.2 IU/ml, p = 0.04 and 0.4 vs. 1.6, p <0.01), less positive (50 vs. 86%, p = 0.03 and 48 vs. 75%, p <0.01) and more false negative (45 vs. 0%, p <0.01 and 26 vs. 11%, p = 0.04) QFT results. In Tanzanian patients, logistic regression analysis adjusted for sex, age, HIV and alcohol consumption showed an association of smoking with false negative (OR 17.1, CI: 3.0-99.1, p <0.01) and indeterminate QFT results (OR 5.1, CI: 1.2-21.3, p = 0.02). CONCLUSIONS: Cigarette smoking was associated with false negative and indeterminate IGRA results in both a high and a low TB endemic setting independent of HIV status.