We observed that the human CD40 ligand (CD40L) gene 5′-flanking region conferred weak promoter activity in activated CD4 T cells, suggesting that additional regions are required for optimal CD40L gene transcription. We therefore examined a 3′-flanking segment of the CD40L gene, which contained a putative NF-κB/Rel ciselement, for its ability to enhance CD40L promoter function. This segment augmented CD40L promoter activity in an orientation-independent manner in CD4 T-lineage cells but not in human B cell or monocyte cell lines. Mapping of CD4 T-lineage cell nuclei identified a DNase I-hypersensitive site in the flanking region near the NF-κB/Rel sequence, suggesting a transcriptional regulatory role. This was further supported by truncation analysis and site-directed mutagenesis, which indicated that the CD40L 3′-flanking NF-κB/Rel cis-element was critical for enhancer function. Electrophoretic mobility shift assays showed that the cis-element preferentially bound the p50 form of the NF-κB1 gene contained in human T cell nuclear protein extracts. This binding also appeared to occur in vivo in CD4 T cells based on chromatin immunoprecipitation assays using NF-κB p50-specific antiserum. Together, these results suggest that the CD40L gene 3′-flanking region acts as a T cell-specific classical transcriptional enhancer by a NF-κB p50-dependent mechanism.