Osmoprotective genes are tonicity-activated genes involved in cellular osmoadaptation to hypertonicity and considered to be regulated by a specific transcription factor called tonicity-responsive enhancer-binding protein (TonEBP). In the brain we had previously established that TonEBP was expressed and tonicity-induced in neurons only. Here we have compared in various brain regions of rats subjected to systemic hypertonicity, the cellular expression of TonEBP through immunocytochemistry and the cellular expression of osmoprotective genes, namely aldose reductase (AR), sodium-dependent myo-inositol transporter (SMIT), betaine/GABA transporter (BGT1) and taurine transporter (TauT), by in situ hybridization using non-radioactive digoxigenin-labeled riboprobes. In neurons where TonEBP was strongly tonicity-induced, AR-mRNA labeling was strongly increased in some subsets (e.g. hippocampus pyramidal cells, cerebellar Purkinje cells and neurons of the hypothalamic magnocellular nuclei) but remained undetectable in some other subsets (e.g. neurons in cerebral cortex). Tonicity-induced AR-mRNA labeling was observed only several hours after the tonicity-induced expression of TonEBP. SMIT-mRNA labeling was tonicity-induced as densely and evenly distributed dots in neuron poor regions (e.g. cerebral cortex layer I and hippocampus stratum lacunosum-moleculare). The tonicity-induced expression of SMIT-mRNA may thus occur in non-neuronal cells, presumably astrocytes, where TonEBP is neither significantly expressed, nor tonicity-induced. In neurons showing a strong tonicity-induced expression of TonEBP, no SMIT-mRNA labeling was observed. BGT1-mRNA and TauT-mRNA labeling could not be detected, even after systemic hypertonicity. The present work reveals large discrepancies between the cellular distribution of the tonicity-induced expression of osmoprotective genes and that of their regulatory transactivator TonEBP. Depending on the cell subsets and the osmoprotective genes, TonEBP may appear insufficient or conversely unnecessary for the tonicity-induced activation of an osmoprotective gene. Altogether our results show that brain cells, even from the same class, activate distinct osmoprotective genes through distinct activation processes to adapt to hypertonicity.