Interleukin (IL)-12 is a heterodimeric cytokine consisting of the p40 and p35 chains encoded on separate chromosomes. Coordinated expression of the two constituent genes is crucial for appropriate immune responses in timing, location, and magnitude. Interferon (IFN)-γ priming of IL-12 production by macrophages represents an important physiological process in vivo for escalated cellular response to microbial infections. We provide evidence that IFN regulatory factor (IRF)-1–deficient macrophages have a selective impairment in mRNA synthesis of IL-12 p35 but not the p40 gene, and a strong deficiency in the production of IL-12 p70 but not p40. We demonstrate that the levels of IL-12 p35 protein stimulated by IFN-γ and lipopolysaccharide (LPS) correspond to those of its mRNA, and that the nuclear factor κB signaling pathway is essential for the induction of IL-12 p35 transcription by LPS. IRF-1 plays a major role in the transcriptional activation of the IL-12 p35 gene, but not of the p40 gene, by physically interacting with an inverted IRF element within the IL-12 p35 promoter upon IFN-γ activation. Moreover, IRF-1–mediated transcriptional activation of the p35 promoter requires the cooperation of two adjacent Sp1 elements. Thus, IRF-1 acts as a critical component of IFN-γ signaling in the selective activation of IL-12 p35 transcription in synergy with LPS-mediated events.