Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is the world's leading cause of death in humans from a single infectious agent. A safe and effective vaccine against this scourge is urgently needed. This study demonstrates that immunization with the 30-kDa major secretory protein, alone or in combination with other abundant extracellular proteins of M. tuberculosis, induces strong cell-mediated immune responses and substantial protective immunity against aerosol challenge with virulent M. tuberculosis bacilli in the highly susceptible guinea pig model of pulmonary tuberculosis. Protection is manifested by decreased clinical illness including decreased weight loss, reduced mortality, and decreased growth of M. tuberculosis in the lungs and spleens of immunized animals compared with sham-immunized controls. This study demonstrates that purified major extracellular proteins of M. tuberculosis are candidate components of a subunit vaccine against tuberculosis and provides compelling support for the concept that extracellular proteins of intracellular pathogens are key immunoprotective molecules.