NF-kappaB/Rel transcription factors, implicated in inflammatory and immune responses against pathogens, are regulated by IkappaB proteins. The physiological and molecular function of the IkappaB family member Bcl-3 is understood only poorly. In this study, the role of Bcl-3 in an innate immune response was examined by gene targeting. We demonstrate that Bcl-3(-/-) mice are highly susceptible to Listeria monocytogenes infection. This correlates with diminished production of IL-12 p70 and IFN-gamma in vivo, which is mainly due to elevated synthesis of IL-10. Isolated peritoneal macrophages from Bcl-3(-/-) mice also produce elevated amounts of IL-10, which inhibit IL-12 p70 synthesis in an autocrine fashion. Thus, these data establish Bcl-3 as an inhibitor of IL-10 expression in macrophages. Furthermore, we show that Bcl-3 is not implicated in IL-10 mRNA stabilization but regulates the initiation of IL-10 transcription. Taken together, our results show that an essential function of Bcl-3 during an innate immune response against bacteria is to inhibit transcription of the IL-10 gene in macrophages.