Compared to other organs the cells of the renal inner medulla are challenged with an environment of highly variable osmolality mediated mainly by marked increases in Na⁺ and urea. We performed gene expression profiling using primary cultured rat renal inner medullary collecting duct cells which were cultivated for 6 days at 300, 600 or 900 mosmol/kg to identify novel signalling pathways that are affected by osmolality. Differentially expressed genes were identified using the BRB Array Tools and functional molecular network were identified with Ingenuity Pathway Analysis. Comparing cells grown in 300 vs. 600 mosmol/kg, more than 1600 genes and vs. 900 mosmol/kg more than 3300 genes were differentially expressed. Pathway analysis showed that genes involved in the WNT/b-catenin (Ctnnb) signalling were down regulated due to hypertonicity. Western blot analysis confirmed the down regulation also on the protein level. We observed a decrease in protein expression for example for WNT5a, LRP6, DVL3 and Ctnnb. Hypertonicity reduced the expression of Ctnnb which was independent from the action of the glycogen synthase kinase-3. Also the intracellular localization of Ctnnb was altered by osmolality changes. While Ctnnb showed diffuse distribution at 300 mosmol/kg at 600 or 900 mosmol/kg it accumulated at the plasma membrane and in part also in the nucleus. Similar differences were observed for the phosphorylated Ctnnb protein. In conclusion the adaptation to a higher tonicity is associated with massive changes in gene expression. Among these changes in expression of members involved in the WNT/ß-catenin pathway and localization of Ctnnb was evident indicating a prominent function of this pathway for the osmoadaptation.