Among various inflammatory mediators produced at the early stage of infection, TH1 cytokines such as interferon-γ, interleukin 12 (IL-12) and IL-18 play a pivotal role for development of protective T cells. It is further known that the production of these cytokines is induced by viable, but not killed bacteria, which are capable of generating protective immunity. To identify the mycobacterial factor contributing to the TH1 cytokine productions, we prepared a culture filtrate from 1d-culture of viable BCG (vBCG) and measured the cytokine-inducing activity. The culture filtrate elicited IL-12p40 production from peritoneal macrophages. However, the similar preparation derived from killed BCG did not induce the cytokine production. The cytokine-inducing activity was detected in macrophages of C3H/HeJ mice and was not affected by E5531, LPS antagonist. Furthermore, the culture filtrate did not induce NF-κB activation in HEK293 cells transfected with TLR4 and MD-2. However, activation of the transcription factor was induced in cells transfected with TLR2 by stimulation with vBCG-derived culture filtrate. The cytokine-inducing activity was resistant to treatment with DNase and partially sensitive to treatment with proteinase K, suggesting that some proteinaceous and non-proteinaceous factors are involved in the cytokine production. The similar activity was also detected in the culture filtrate obtained from viable, but not killed M. tuberculosis H37Rv. These results suggest that the early secreted components from viable mycobacteria activate macrophages to produce IL-12 via TLR2 dependent pathway. It is likely that this is a critical interaction between bacteria and macrophages for the generation of protective immunity.