Peritoneal exudate cells of mice were stimulated with a streptomycin-dependent Mycobacterium tuberculosis strain, 18b. Gamma interferon production by natural killer cells depending on interleukin-12 and interleukin-18 was induced only in the presence of a high dose of streptomycin. This study suggested the requirement of active bacterial metabolism for this host response.Mycobacterium tuberculosis is a causative agent of tuberculosis in human being. One-third of the world's population is currently infected with tubercle bacilli, and the global incidence of active tuberculosis per year is around 8 million cases (2). It is of great importance to understand the basic mechanism for the induction of the immune response of the host against M. tuberculosis. The generation of protective immunity against tuberculosis is dependent on the induction of CD4 ϩ Th1 cells capable of producing gamma interferon (IFN-␥) upon stimulation with specific antigen. IFN-␥ contributes to the development of acquired resistance via the activation of macrophages (3). The importance of IFN-␥ in the resistance of mice to M. tuberculosis has been confirmed by utilizing IFN-␥ knockout mice and IFN-␥ receptor knockout mice (1, 4, 11). IFN-␥ is crucial also for the development of protective T cells. In our previous study, the treatment of mice with anti-IFN-␥ antibody during primary immunization with viable cells of M. bovis bacillus Calmette-Guérin reduced the number of antigen-specific IFN-␥-producing cells and abolished the generation of protective immunity (26). Thus, IFN-␥ is indispensable for both induction and expression of protective immunity against tuberculosis.It has been shown that CD4 ϩ protective T cells are generated after infection with a sublethal dose of M. tuberculosis or M. bovis bacillus Calmette-Guérin, whereas such effector T cells are hardly induced by immunization with killed bacteria (14). We have found that the failure of killed bacteria to induce effective protective immunity in mice is due to the absence of an IFN-␥-inducing ability that is observed exclusively in viable bacilli (25,26). Killed M. tuberculosis has been prepared generally by treatment with heating, germicides, or irradiation (7,16,20,24), but such treatment may affect several bacterial components physically or chemically. In order to address whether the significant difference in the IFN-␥-inducing abilities of viable and killed M. tuberculosis is due to some undesirable changes introduced during the killing process or actually due to the viability itself, we have employed a streptomycin (SM)-dependent M. tuberculosis strain, 18b, in this study.This particular strain, originally isolated in 1955 (6), has been maintained as a stock for a long time at the National Institute of Infectious Diseases in Japan, so we first confirmed whether this strain maintained the original SM dependency. On a Middlebrook 7H10 agar plate, strain 18b never grew during 5 weeks of observation in the absence of SM (Fig. 1). However, the addition of SM at concentrations of 50 g/ml...