MPT63 (Rv1926c), a major secreted protein of Mycobacterium tuberculosis, is immunoreactive in antibody assays in humans and animals and provides protection as a combined DNA vaccine in mice. This study was undertaken to determine the reactivity of MPT63 in T helper 1 (Th1) cell assays, i.e. antigen-induced proliferation and interferon-gamma secretion, using peripheral blood mononuclear cells (PBMCs) obtained from 72 Mycobacterium bovis Bacille Calmette-Guérin vaccinated healthy subjects. PBMCs were tested with complex mycobacterial antigens and pools of synthetic peptides corresponding to MPT63, MPB70, MT24, PPE68, CFP10 and ESAT-6. The results showed that MPT63 induced moderate Th1 cell reactivity which was equivalent to the reactivity induced by other secreted antigens of M. tuberculosis, i.e. MT24 and MPB70. Furthermore, human leucocyte antigen (HLA) heterogeneity of the responding donors suggested that MPT63 was presented to Th1 cells promiscuously. Analysis of the MPT63 sequence and its peptides for binding to 51 alleles of 9 serologically defined HLA-DR molecules, using a virtual matrix-based prediction program (ProPred) showed that MPT63 sequence could bind to all the 51 alleles, whereas 9 of the 10 peptides of MPT63 were also predicted to bind promiscuously. When tested with PBMCs of HLA-DR heterogeneous donors that responded to MPT63 in interferon-gamma assays, at least 9 of the 10 peptides of MPT63 were recognized by PBMCs from HLA heterogeneous donors. These results suggested that promiscuous Th1 cell reactive epitopes are scattered throughout the sequence of MPT63, and further support the inclusion of this protein in an antigen cocktail to develop a new anti-tuberculosis vaccine.