Tuberculosis continues to be a major disease and the single greatest infectious cause of human mortality. Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been cloned and are being exploited in the development of improved vaccines and diagnostic reagents. One of these antigens, the 16-kDa antigen, shows sequence homology with alpha-crystallin related small heat shock proteins. We have cloned and expressed the 16-kDa antigen also known as Hsp16-3. Site-directed spin labeling was used to explore the symmetry of the oligomeric structure. Hsp 16-3 consists of a trimer of trimers assembled by subunit interactions in the highly conserved c-terminal domain.