The sequencing of the Mycobacterium tuberculosis genome revealed the existence of several genes encoding novel proteins with unknown functions, one of which is the proline-threonine repetitive protein (PTRP; Rv0538). Genomic studies of various mycobacterial species and M. tuberculosis clinical isolates demonstrate that ptrp is specific to the M. tuberculosis complex and ubiquitous in clinical isolates. Enzyme-linked immunosorbent assay, Western blot analysis, and electron microscopic evaluation of M. tuberculosis subcellular fractions and intact bacteria confirm that PTRP is a cell wall protein. Antibodies to PTRP are present in serum specimens from human immunodeficiency virus (HIV)-negative, tuberculosis (TB)-positive and HIV-positive, TB-positive patients but not purified protein derivative (PPD)-negative or PPD-positive healthy control subjects, demonstrating its diagnostic potential. Epitope mapping of PTRP delineated 4 peptides that can identify >80% of sputum smear-positive and >50% of smear-negative, HIV-negative, TB-positive patients and >80% of HIV-positive, TB-positive patients. These results demonstrate that immunodominant epitopes of carefully selected M. tuberculosis-specific proteins can be used to devise a simple peptide-based serodiagnostic test for TB.