We have previously presented evidence demonstrating that mice deficient in NF-kappaB subunits are susceptible to colitis induced by the pathogenic enterohepatic Helicobacter species, H. hepaticus. However, it has not been determined whether NF-kappaB is required within inhibitory lymphocyte populations, within cells of the innate immune system, or both, to suppress inflammation. To examine these issues, we have performed a series of adoptive transfer experiments using recombination-activating gene (Rag)-2(-/-) or p50(-/-)p65(+/-)Rag-2(-/-) mice as hosts for wild-type (WT) and p50(-/-)p65(+/-) lymphocyte populations. We have shown that although the ability of H. hepaticus to induce colitis in Rag-2(-/-) mice is inhibited by the presence of either WT or p50(-/-)p65(+/-) splenocytes, these splenocyte populations are unable to suppress H. hepaticus-induced colitis in p50(-/-)p65(+/-)Rag-2(-/-) mice. Colitis in these animals is characterized by increased expression of inflammatory cytokines including IL-12 p40, and depletion of IL-12 p40 from p50(-/-)p65(+/-) mice ameliorates H. hepaticus-induced disease. Consistent with a primary defect in the regulation of IL-12 expression, H. hepaticus induced markedly higher levels of IL-12 p40 in p50(-/-)p65(+/-) macrophages than in WT macrophages. These results suggest that inhibition of H. hepaticus-induced IL-12 p40 expression by NF-kappaB subunits is critical to preventing colonic inflammation in response to inflammatory microflora.