Transcription of germline Ig constant region genes and associated switch regions is an early and essential step in heavy chain class switch recombination. Transcription of the germline Cgamma1 and C epsilon Ig genes is induced by IL-4 via STAT6 activation; CD40 signaling can independently induce transcription of these genes and act in synergy with IL-4 to increase expression. In the present study, we investigated the role of three tandem NF-kappaB sites (site 1, -95; site 2, -71; site 3, -53) in the regulation of the germline Cgamma1 Ig promoter by CD40 Ligand (CD40L) and IL-4 in the mouse B lymphoma cell line, BCL1-3B3. Germline gamma1 transcripts are induced by CD40L and by IL-4 in BCL1-3B3 and the combination of signals is synergistic, as in normal B cells. EMSA with crude nuclear extracts demonstrated that stimulation with CD40L results in the induction of NF-kappaB complexes that bind to each of the three NF-kappaB sites and are composed mainly of p50 and RelB, but also include c-Rel and p65. Surprisingly, site-specific mutagenesis of the NF-kappaB sites did not reduce CD40-responsiveness of germline gamma1 promoter-luciferase reporter constructs transiently transfected into BCL1-3B3. Mutation in any one NF-kappaB site, however, significantly reduced overall transcriptional activity of the promoter, both basal and induced, suggesting a role in basal promoter function. In addition, activation of the promoter by IL-4 was blocked by mutation of all three NF-kappaB sites and similarly reduced by mutation of site 1, suggesting that NF-kappaB-STAT6 interactions may be necessary for STAT6-mediated transactivation of the germline gamma1 promoter. The results suggest that the three NF-kappaB sites may serve as a focus for formation of a higher-order transcription complex including STAT6, NF-kappaB and components of the basal transcription apparatus.