The enhancer complex located 3' to the C(H)alpha gene in the IgH locus (3alphaE) may regulate B cell function through its ability to act as a locus control region. Multiple, functionally relevant NF-kappaB binding sites are located within the 3'alphaE. NF-kappaB subunits, especially p50 and c-Rel, have also been shown to play critical and differential roles in regulating B cell proliferation, Ig secretion, germline C(H) transcription and Ig class switching. Thus, NF-kappaB could regulate B cell function in part through modulation of 3'alphaE activity. In this study we determined whether p50 and/or c-Rel regulate 3'alphaE activity in normal murine B cells and whether this depends on the nature of the B cell activator. For this purpose, we crossed p50- and c-Rel-deficient mice with mice that are transgenic for a 3'alphaE-hsl,2-human beta-globin reporter gene, and established p50(-/-) or c-Rel(-/-) mice homozygous for the enhancer transgene. We show, using optimal stimulating conditions, that p50 selectively augments 3'alpha E-hsl,2 activity in lipopolysaccharide-activated B cells, whereas c-Rel is required for optimal 3'alphaE-hs1,2 induction in B cells activated through CD40.