Tuberculosis (TB) serological testing with antigen complexes, although very sensitive, is not always as specific due to reactive serum antibodies in patients with inactive TB or nontuberculous infections. Since the use of recombinant M. tuberculosis proteins may enhance specificity, this study was designed to evaluate a novel 34 kDa tuberculosis complex-specific protein as a component of an antigen panel of recombinant proteins. Seventy patients with active TB (41 positive and 29 negative for acid-fast bacilli (AFB) in sputum) were evaluated, in comparison with 30 tuberculin purified protein derivative skin test positive (PPD+) and 30 PPD- normals, 20 subjects with inactive TB and 20 PPD+ subjects with nontuberculous pneumonia as controls. Serum antibody levels were quantified using enzyme linked immunosorbent assay (ELISA) tests with MS2-34, a fusion protein comprising the NH2-terminal 16 kDa of the 34 kDa protein, a recombinant 38 kDa protein (p38), and PPD. Using MS2-34 and p38 as an antigen panel in active TB patients yielded higher sensitivity and negative predictive value (sensitivity 86%; negative predictive value 91%) than using PPD (sensitivity 66%; negative predictive value 81%). Importantly, the MS2-34+p38 panel yielded a higher sensitivity (83%) than PPD (66%) in the subset of AFB- active TB patients. Thus, this novel protein increases sensitivity and specificity of serological testing for TB when used in panels of recombinant proteins.