Interleukin-12 (IL-12) and IL-23 (which share a p40 subunit) are pivotal cytokines in the generation of protective Th1/Th17-type immune responses upon infection with the intracellular pathogen Mycobacterium tuberculosis. The role of IL-12 and IL-23 in protection conferred by the tuberculosis vaccine Mycobacterium bovis bacillus Calmette-Guerin (BCG) is, however, less well documented. By using an autovaccine approach, i.e., IL-12p70 cross-linked with ovalbumin and PADRE peptide formulated with the GSK proprietary adjuvant system AS02(V), we could specifically neutralize IL-12 while leaving the IL-23 axis intact. Neutralization of IL-12 before M. tuberculosis challenge rendered C57BL/6 mice highly susceptible, resulting in 30-fold-higher CFU in spleen and lungs and accelerated mortality. In contrast, neutralization of IL-12 in BCG-vaccinated mice prior to M. tuberculosis challenge only marginally affected vaccine-mediated protection. Analysis of cytokine production in spleen and lungs 3 weeks post-TB challenge by enzyme-linked immunosorbent assay and functional and flow cytometric assays showed significantly reduced mycobacterium-specific gamma interferon (IFN-gamma) responses in M. tuberculosis-infected and BCG-vaccinated mice that had been treated with the autovaccine. Purified protein derivative-induced tumor necrosis factor alpha (TNF-alpha), IL-6, and IL-17A levels, however, were highest in lungs from BCG-vaccinated/IL-12-neutralized animals, and even unstimulated lung cells from these mice produced significant levels of the three cytokines. Mycobacterium-specific IL-4 and IL-5 production levels were overall very low, but IL-12 neutralization resulted in increased concanavalin A-triggered polyclonal secretion of these Th2-type cytokines. These results suggest that TNF-alpha, IL-6, and IL-17A may be more important pulmonary effector molecules of BCG-mediated protection than IFN-gamma in a context of IL-12 deficiency