Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays an important role in a variety of infectious and autoimmune disorders. Its transcription is regulated in a stimulus- and cell-type-specific manner via the recruitment of distinct DNA/activator complexes forming secondary structures or enhanceosomes. NFATp, a member of the nuclear factor of activated T cells (NFAT) family of transcription factors, plays a critical role in TNF gene regulation under a variety of conditions. In this study, we show that NFAT5, the most recently described NFAT family member, binds to the TNF promoter in a manner distinct from other NFAT proteins and is a key mediator in the activation of TNF gene transcription during hypertonic stress alone.