The molecular basis of the immunomodulatory properties of 1α, 25-dihydroxyvitamin D 3 (1α, 25 (OH) 2 D 3) remains elusive. We demonstrate here that 1α, 25 (OH) 2 D 3-mediated suppressive effects on the inducible expression of cytokine genes in human T cells may, in part, be due to diminished activity of the transcription factor NFAT. The vitamin D 3 receptor (VDR) and its heterodimeric partner retinoid X receptor α (RXRα) specifically bound to the distal NFAT site in the human IL-2 promoter, and this binding was abolished by mutating unique regions in the NFAT oligonucleotide. In vitro inhibition of NFAT complex formation was noted when VDR-RXRα heterodimers were added to DNA binding reactions containing nuclear extracts from activated B or T cells, whereas in vitro NFκB complex formation was not significantly influenced. Furthermore, 1α, 25 (OH) 2 D 3 treatment of activated T cells resulted in decreased formation of NFAT complexes detected upon incubation of nuclear extracts from these cells with 32 P-labeled probe. Transient expression of both VDR and RXRα, but not of a single component, was capable of inhibiting expression of a NFAT-driven reporter gene in stimulated Jurkat cells in a ligand-dependent manner. These results suggest that NFAT plays a crucial role in 1α, 25 (OH) 2 D 3-mediated immunosuppressive activity.