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In desert rodents, the production of concentrated urine is essential for survival in xeric environments in order to conserve water. Reabsorption of water in the kidney is dependent on large osmotic gradients in the renal medulla. This causes the renal cells to be bathed in a hypertonic extracellular fluid that can compromise cellular function. In response to hypertonicity, kidney cells accumulate compatible, non-ionic osmolytes that lower the ionic strength within the cells to isotonic levels by replacing intracellular ionic electrolytes. The tonicity-responsive enhancer binding protein ( TonEBP) is a transcription factor that regulates the expression of genes that encode proteins that catalyse the accumulation of compatible osmolytes. We investigated the expression of TonEBP mRNA and protein and compatible osmolyte genes in the Spinifex hopping mouse, Notomys alexis, an Australian desert rodent that produces a highly concentrated urine. TonEBP mRNA expression was unchanged after 3 days of water deprivation but was significantly increased after 7 and 14 days of water deprivation. Immunohistochemistry showed that during water deprivation TonEBP had translocated from the cytoplasm into the nucleus of cells in the renal medulla and papilla. In addition, 3, 7 and 14 days of water deprivation caused a significant increase in aldose reductase ( AR), myo-inositol ( SMIT), betaine/GABA ( BGT-1) and taurine ( TauT) transporter mRNA expression, which is indicative of an increase in TonEBP activity. In desert rodents, TonEBP regulation of gene transcription is probably an important mechanism to protect renal cells in the face of the large corticomedullary gradient that is required to concentrate urine and conserve water.