CD95(APO‐1 / Fas) ligand (CD95L) gene expression is critically involved in activation‐induced T cell apoptosis. We and other have previously shown that HIV‐1 Tat which is essential for efficient HIV gene expression sensitizes CD95‐mediated apoptosis and up‐regulates CD95L expression in T cells. In the present study we have investigated the regulatory mechanism for CD95L expression. Two NF‐κB binding sites are localized at − 537 to − 521 and − 57 to − 47 (relative to the transcription start site) of the human CD95L promoter. We show that both elements bind to NF‐κB and SP‐1 transcription factors and NF‐κB is involved in transactivation of the CD95L promoter upon T cell activation. Mutations at each NF‐κB site by two base pair substitutions resulted in 30 – 70 % reduction of the promoter activity. The effect of Tat on the human CD95L promoter activity was mapped to the same sites. Mutation of each NF‐κB site also impaired the effect of Tat on CD95L promotor activity. We also show that ectopic expression of Tat protein in Jurkat T cells greatly increases NF‐κB binding to its target DNA. Our studies provide evidence that Tat‐enhanced CD95L expression is regulated at least in part by the NF‐κB sites of the promoter.