Identification of M. tuberculosis ESAT-6 proteins has enhanced research for developing new specific and reliable in vitro diagnostic tests for pulmonary tuberculosis. The aim of this study was to assess the diagnostic potential of ESAT-6 synthetic overlapping peptides mixture (Pepmix) for diagnosis of pulmonary M. tuberculosis infection in Egypt. IFN-gammaand IL-17 production was assessed in whole-blood culture assay after overnight stimulation with M. tuberculosis crude culture filtrate (CF), purified 38 kDa antigen, and ESAT-6 Pepmix. Concentration of IFN-gamma and IL-17 was evaluated in the culture supernatant by ELISA. Patients with active pulmonary TB patients (n = 37), Tuberculin Skin Test (TST) positive healthy subjects (n = 16) and TST negative healthy subjects (n = 16) were included. Results indicated that IFN-gammaproduction following stimulation with crude CF, and 38 kDa purified antigen was not significantly different between patients and TST+ healthy controls. In contrast, Pepmix clearly distinguished between patients and TST+ healthy subjects with sensitivity of 70.3 % and specificity 96.9 %. The concentration of secreted IL-17 was significantly lower than IFN-gamma, and no statistically significant difference was found between groups. It is concluded that IFN-gamma assay using ESAT-6 Pepmix, as antigenic stimulus, has the potential to rapidly diagnose pulmonary TB infection.