Immune responses to Mycobacterium tuberculosis are analyzed in mice which have been immunized with Mycobacterium vaccae to examine novel ways of altering protective immunity against M. tuberculosis. The spleen cells of mice immunized with M. vaccae proliferate and secrete gamma interferon (IFN-gamma) in response to challenge with live M. tuberculosis in vitro. Immunization with M. vaccae results in the generation of CD8+ T cells which kill syngeneic macrophages infected with M. tuberculosis. These effector cytotoxic T cells (CTL) are detectable in the spleen at 2 weeks after immunization with M. vaccae but cannot be found in splenocytes 3 to 6 weeks postimmunization. However, M. tuberculosis-specific CTL are revealed following restimulation in vitro with heat-killed M. vaccae or M. tuberculosis, consistent with the activation of memory cells. These CD8+ T cells secrete IFN-gamma and enhance the production of interleukin 12 when cocultured with M. tuberculosis-infected macrophages. It is suggested that CD8+ T cells with a cytokine secretion profile of the Tc1 class may themselves maintain the dominance of a Th1-type cytokine response following immunization with M. vaccae. Heat-killed M. vaccae deserves attention as an alternative to attenuated live mycobacterial vaccines.