Engagement of CD40 by its ligand CD154 induces IL-6 production by B lymphocytes. We previously reported that this IL-6 production is dependent upon binding of the adapter protein TNF receptor-associated factor 6 to the cytoplasmic domain of CD40, while binding of TNF receptor-associated factors 2 and 3 is dispensable, as is the activation-induced nuclear translocation of NF-κB. The present study was designed to characterize CD40-mediated transcriptional control of the IL-6 gene in B cells. CD40 engagement on B lymphocytes activated the IL-6 promoter, and mutations in the putative binding sites for AP-I and C/EBP transcription factors reduced this activation. Interestingly, a mutation in the putative NF-κB binding site completely abrogated the basal promoter activity, thus also rendering the promoter unresponsive to CD40 stimulation, suggesting that this site is required for binding of NF-κB constitutively present in the nucleus of mature B cells. The expression of dominant negative Fos or C/EBPα proteins, which prevent binding of AP-1 or C/EBP complexes to DNA, also reduced CD40-mediated IL-6 gene expression. Furthermore, CD40 stimulation led to phosphorylation of c-Jun on its activation domain, implicating CD40-mediated Jun kinase activation in the transcriptional regulation of IL-6 production.