IL-12p35−/− p40−/− mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis infection. In this study IL-12p35−/− mice, which are able to produce endogenous IL-12p40, cleared M. bovis BCG and showed reduced susceptibility to pulmonary M. tuberculosis infection, which was in striking contrast to the outcome of mycobacterial infection in IL-12p35−/− p40−/− mice. Resistance in wild-type and IL-12p35−/− mice was accompanied by protective granuloma formation and Ag-specific delayed-type hypersensitivity responses, which were impaired in susceptible IL-12p35−/− p40−/− mice. Furthermore, IL-12p35−/− mice, but not IL-12p35−/− p40−/− mice, mounted Ag-specific Th1 and cytotoxic T cell responses. In vivo therapy with rIL-12p40 homodimer restored the impaired delayed-type hypersensitivity responses in M. bovis BCG-infected IL-12p35−/− p40−/− mice and reverted them to a more resistant phenotype. Together, these results show evidence for a protective and agonistic role of endogenous and exogenous IL-12p40 in mycobacterial infection, which is independent of IL-12p70.