The transcription factor nuclear factor of activated T cell 5 (NFAT5) is activated by the stress of hypertonicity (e.g., high NaCl). Increased expression of NFAT5 target genes causes accumulation of protective organic osmolytes and heat shock proteins. Under normotonic conditions (∼300 mosmol/kgH2O), NFAT5 is distributed between the nucleus and cytoplasm, hypertonicity causes it to translocate into the nucleus, and hypotonicity causes it to translocate into the cytoplasm. The mechanism of translocation is complex and not completely understood. NFAT5-T298 is a known contact site of NFAT5 with its specific DNA element [osmotic response element (ORE)]. In the present study, we find that mutation of NFAT5-T298 to alanine or aspartic acid not only reduces binding of NFAT5 to OREs (EMSA) but also proportionately reduces high NaCl-induced nuclear translocation of NFAT5. Combined mutation of other NFAT5 DNA contact sites (R293A/E299A/R302A) also greatly reduces both specific DNA binding and nuclear localization of NFAT5. NFAT5-T298 is a potential phosphorylation site, but, using protein mass spectrometry, we do not find phosphorylation at NFAT5-T298. Further, decreased high NaCl-induced nuclear localization of NFAT5 mutated at T298 does not involve previously known regulatory mechanisms, including hypotonicity-induced export of NFAT5, regulated by phosphorylation of NFAT5-S155, XPO1 (CRM1/exportin1)-mediated export of NFAT5 from the nucleus, or hypertonicity-induced elevation of NUP88, which enhances nuclear localization of NFAT5. We conclude that specific DNA binding of NFAT5 contributes to its nuclear localization, by mechanisms, as yet undetermined, but independent of ones previously described to regulate NFAT5 distribution.