CD40 ligand (CD40L, CD154) is a T cell cytokine with highly regulated expression that requires the transcription factor nuclear factor of activated T cells (NF-AT) to bind at two sites in the proximal CD40L promoter. We have determined that the distal CD40L promoter (-500 to -1300 bp from start of transcription) conveys superior promoter activity in reporter gene assays. Within the distal promoter, we have identified a third NF-AT binding site, at -761 to -756. Oligonucleotides incorporating each of the three NF-AT sites cross-compete for binding of nuclear extracts from activated T cells and bind NF-ATc2 by antibody supershift. Mutation of the distal NF-AT site reduces activity of the 1300 bp CD40L promoter construct to that of the proximal 500 bp construct, which includes only two NF-AT sites. This suggests that the newly identified NF-AT site is the major mediator of transcriptional activation in the distal CD40L promoter,