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Members of the nuclear factor of activated T cells (NFAT) are involved in the induction of a number of cytokine genes. We report here cDNA cloning and chromosomal localization of a murine homologue of human NFATx, designated as mNFATx1, and its splicing variants mNFATx2 and m delta NFATx. Northern blot analysis showed mNFATx1 to be predominantly expressed in the thymus. mNFATx1, but not m delta NFATx, produced in COS-7 cells, bound to all NFAT-binding sites of the interleukin (IL)-2 and IL-4 promoters tested. Immunofluorescence assay showed that both mNFATx1 and m delta NFATx introduced into COS-7 cells localized predominantly to the cytoplasm, but did translocate to the nucleus, either by cotransfection with an active form of calcineurin or wild-type calcineurin followed by stimulation with calcium ionophore. Translocation of mNFATx1 correlated well with activation of the murine IL-2 promoter; mNFATx1 translocated under conditions described above, in combination with phorbol 12-myristate 13-acetate, activated the transiently transfected murine IL-2 promoter. Thus, nuclear-translocated mNFATx1 is involved in activation of the IL-2 promoter. These results provide the first evidence for the requirement of calcineurin in the control of mNFATx imported from the cytoplasm to the nucleus and implies that mNFATx may possibly be a substrate of calcineurin in vivo.