In recent years, there has been considerable focus on the discovery and characterization of proteins derived from Mycobacterium tuberculosis leading to the identification of a number of candidate antigens for use in vaccine development or for diagnostic purposes. Previous experiments have demonstrated an important immunological role for proteins encoded by the RD1 region, which is absent from all strains of bacillus Calmette-Guérin (BCG) but present in the genomes of virulent M. bovis and M. tuberculosis. Herein, we have studied human T-cell responses to the antigen encoded by the putative open reading frame (rv3878) of the RD1 region. Immunoblot analysis revealed that rv3878 was expressed and the native protein was designated TB27.4. Immunological evaluations demonstrate that TB27.4 elicits a prominent immune response in human tuberculosis patients with a dominant region in the C-terminal part of the molecule. In contrast, very limited responses were seen in M. bovis BCG-vaccinated donors. This study therefore emphasizes the diagnostic potential of proteins encoded by the RD1 region.