Interleukin 12 (IL-12) is an inducible cytokine composed of 35-and 40-kDa subunits that is critical for promoting T helper type 1 development and cell-mediated immunity against pathogens. The 40-kDa subunit, expressed by activated macrophages and B cells, is induced by several pathogens in vivo and in vitro and is augmented or inhibited by gamma interferon (IFN-γ) or IL-10, respectively. Control of IL-12 p40 expression is therefore important for understanding resistance and susceptibility to a variety of pathogens, including Leishmania major and perhaps human immunodeficiency virus. In this report, we provide the first characterization of IL-12 p40 gene regulation in macrophages. We localize inducible activity of the promoter to the sequence 2 122 GGGGAATTTTA 2 132 not previously recognized to bind Rel family transcription factors. We demonstrate binding of this sequence to NF-κB (p50/p65 and p50/c-Rel) complexes in macrophages activated by several p40-inducing pathogens and provide functional data to support a role for NF-κB family members in IL-12 p40 activation. Finally, we find that IFN-γ treatment of cells enhances this binding interaction, thus potentially providing a mechanism for IFN-γ augmentation of IL-12 production by macrophages.