We have previously shown that both CD40 ligation and IL-4 induce lymphotoxin alpha (LT alpha) expression in B cells. We generated a series of truncations of the LT alpha upstream region (-915 to +7 bp) and examined their ability to drive expression of a luciferase (LUC) reporter gene in B cells. The CD40-responsive promoter region of LT alpha was mapped to the region spanning -120 to -52 bp. This region contains an NF-kappa B site (-99 to -89 bp) which was shown to form a complex with nucleoproteins from CD40-stimulated B cells that contained the p50/p65 subunits of NF-kappa B. Mutation of the NF-kappa B site within the -356 to +7 bp region of the LT alpha gene completely abolished its capacity to drive transcription of the LUC gene in response to stimulation with CD40, but not to IL-4. The IL-4-responsive promoter region of LT alpha was mapped to the region spanning -265 to -185 bp. This region contains a site for binding to signal transducers and activators of transcription (STAT) proteins (-197 to -189 bp). This site was shown to form a complex with nucleoproteins from IL-4-stimulated B cells that contained STAT6. Mutation of the STAT site within the -356 to +7 bp region of the LT alpha gene completely abolished its capacity to drive transcription of the LUC gene in response to IL-4, but not to anti-CD40. These results demonstrate that CD40 and IL-4 use distinct mechanisms, namely activation of NF-kappa B and STAT6, respectively, to activate transcription of the LT alpha gene in B cells.