The NFAT family transcription factors play crucial roles in immunological and other biological events; however, the functional differences among NFAT members have not been fully elucidated. This study investigated the relative contribution of NFATc2 and NFATc1 to the transactivation of cytokine genes in T cells. Ectopic expression of NFATc2 but not NFATc1, especially its short isoform, enhanced TNF-α synthesis in human T cells at the gene transcription level, whereas both NFATs augmented IL-2 expression. In addition, a reduction of the shortest NFATc1 isoform using RNA interference technology failed to suppress TNF-α expression. The promoter/enhancer activity of the NFAT-binding site in the TNF-α gene was up-regulated by NFATc2 but not by NFATc1, whereas both NFATs associated similarly with this region. A study of mRNA expression using NFATc2/NFATc1 chimeric molecules revealed that the enhancing activity of NFAT on the TNF-α gene was lost by truncation of its C-terminal transactivation domain. In addition, this domain derived from NFATc2 behaved as a dominant negative against the NFAT site in TNF-α promoter-dependent transcriptional activity in T cells. We conclude that the C-terminal transactivation domain in NFAT is crucial for TNF-α gene expression in human T cells.