Macrophages derived from IFN-regulatory factor-1 (IRF-1) and IRF-2 knockout (-/-) and wild-type (+/+) mice were utilized to examine the role of these transcription factors in the regulation of IL-12 mRNA and protein expression. Induction of IL-12 p40 mRNA by LPS was markedly diminished in both IRF-1(-/-) and IRF-2(-/-) macrophages. In contrast, IRF-1(-/-), but not IRF-2(-/-), macrophages exhibited impaired LPS-induced IL-12 p35 mRNA expression. The ability of IFN-gamma to augment LPS-induced IL-12 p40 mRNA further when both stimuli were present simultaneously was significantly diminished in both IRF-1(-/-) and IRF-2(-/-) macrophages, with the most profound impairment observed for IRF-1(-/-) macrophages. Reductions in IL-12 mRNA expression after stimulation with LPS or LPS plus IFN-gamma were accompanied by substantial reductions in IL-12 p40 and IL-12 p70 protein in both IRF-1(-/-) and IRF-2(-/-) macrophages. Priming IRF-1(-/-) and IRF-2(-/-) macrophages with IFN-gamma for 24 h before LPS treatment partially restored impaired IL-12 mRNA and protein production in both IRF-1(-/-) and IRF-2(-/-) macrophages. Depressed IL-12 levels were paralleled by significant reductions in IFN-gamma mRNA expression in IRF-1(-/-) and IRF-2(-/-) macrophages. These results indicate that both IRF-1 and IRF-2 are critical transcription factors in the regulation of macrophage IL-12 and consequently IFN-gamma production.