During antidiuresis, renal medullary cells adapt to their hyperosmotic interstitial environment by increased expression of osmoprotective genes, which is driven by a common transcriptional activator, TonEBP/NFAT5. The present studies addressed the effect of nitric oxide (NO) on expression of osmoprotective genes under hypertonic conditions in MDCK cells. Three structurally unrelated NO-donors dose-dependently blunted tonicity-induced up-regulation of TonEBP/NFAT5 target genes involved in intracellular accumulation of organic osmolytes (aldose reductase, betaine/GABA transporter-1, sodium/myo- inositol transporter), and that of heat shock protein 70. These effects were mediated by reduced transcriptional activity of TonEBP/NFAT5 as assessed by TonEBP/NFAT5-driven reporter constructs. Neither diminished total TonEBP/NFAT5 abundance nor impaired nuclear translocation of TonEBP/NFAT5, the major mechanisms regulating TonEBP/NFAT5 activation, were affected by NO. Furthermore, 8-bromo-cGMP and peroxynitrite failed to reproduce the inhibitory effect of NO, indicating that NO acts directly on TonEBP/NFAT5, rather than through classical NO signalling pathways. These observations disclose a novel inhibitory effect of NO on TonEBP/NFAT5, a finding that may be relevant under pathophysiological settings associated with increased oxidative stress in the renal medulla.