One characteristic of mice prone to a variety of autoimmune diseases is the aberrant regulation of cytokine production by macrophages (Mphi), noted in cells isolated well before the onset of disease. Strikingly, the pattern of IL-12 dysregulation, in particular, is consistent with the nature of the autoimmune disease that will develop in each strain, i.e., elevated in mice prone to Th1-mediated organ-specific disease (nonobese diabetic (NOD) and SJL mice) and reduced in lupus-prone strains (MRL/+ and NZB/W). Mechanistically, the abnormal regulation of IL-12 in these strains was found to be strictly associated with novel patterns of Rel binding in vitro to the unique NF-kappaB site in the IL-12 p40 promoter. In this study, we report several new findings related to these Rel-kappaB interactions. Evaluation of the p40 NF-kappaB site in vivo, assessed by chromatin immunoprecipitation, revealed Rel usage patterns similar to those found in vitro using EMSA, with preferential association of the p40 kappaB site with c-Rel in NOD Mphi but with p50 in NZB/W Mphi. Moreover, blocking c-Rel in primary Mphi, using short interfering RNA, selectively blocked IL-12 production and normalized the minimal, residual IL-12 levels. Nuclear extracts from NOD Mphi were characterized by c-Rel hyperphosphorylation, and dephosphorylation of nuclear proteins completely blocked binding to the kappaB site. In contrast, elevated IkappaB appears to be a likely mechanism accounting for the reduced nuclear c-Rel levels noted in NZB/W Mphi. Alterations in NF-kappaB metabolism thus appear to define a pathway regulating intrinsic IL-12 defects in both diabetes- and lupus-prone strains.