Regulation of interleukin (IL)-12 production by coexpression of tumor necrosis factor (TNF)-α, IL-10, and transforming growth factor (TGF)-β in human monocytes infected with Mycobacterium tuberculosis H37Ra was analyzed. Also, since IL-12 induces interferon (IFN)-γ, the effect of IFN-γ on IL-12 expression was examined. IL-12 mRNA was measured by reverse transcriptase-polymerase chain reaction and IL-12 protein by ELISA. IL-12 p35 mRNA was constitutive and inducible. IL-12 p70 protein paralleled IL-12 p40 protein expression. TNF-α protein expression occurred earlier than IL-12 p40 protein but was not required for IL-12 induction. Addition or neutralization of TGF-β did not significantly alter IL-12 induction. In contrast, recombinant IL-10 reduced IL-12 and neutralization of IL-10 minimally enhanced IL-12. A pronounced increase in IL-12 followed IFN-γ pretreatment, which selectively up-regulated IL-12 p35 mRNA. Further understanding of operative cytokine networks during M. tuberculosis infection may improve strategies for vaccine development and immunotherapy.