Background: The determination of cytokine concentrations in serum and bronchoalveolar lavage fluid (BALF) may contribute to the diagnosis of tuberculosis (TB) since cytokines have been ascribed an important role in TB pathogenesis. Objective: To assess the diagnostic accuracy of TNF-α, IFN-γ and IL-2 levels in serum and BALF of smear-negative pulmonary TB patients. Method: BALF was obtained from the affected lobe in patients with smear-negative TB or other pulmonary diseases (OPD), and from the right middle lobe in healthy controls. ELISA and a nephelometric method were used to detect cytokine and albumin levels. Results: TNF-α levels in BALF were significantly elevated in the TB group (n = 15) compared with the OPD patients (n = 40) and controls (n = 17; p < 0.001). Although these three cytokines correlated well with each other in BALF (p < 0.0001, and r ≧ 0.7, respectively), BALF IL-2 and IFN-γ levels were not significantly different among the groups (p > 0.05). BALF TNF-α or IFN-γ levels were significantly higher in patients with cavitary disease (n = 11) versus those without (n = 61; p < 0.05). However, no significant difference was found between cavitary (n = 7) and non-cavitary TB in cytokine levels (p > 0.05). Neither gender nor smoking status showed any statistical differences in cytokines in the groups (p > 0.05). Sensitivity and specificity of BALF TNF-α were found to be 73 and 76%, respectively. The positive and negative predictive values for BALF TNF-α were 44 and 91%, respectively. Conclusion: In cases of smear-negative TB, BALF TNF-α can be a useful tool to identify healthy subjects rather than smear-negative TB patients.