Genetic studies indicated that mutations of the chloride channel CLC-5 in the kidney are responsible for a group of clinical disorders, collectively called Dent's disease. In the kidney, CLC-5 was found to be expressed in the proximal tubule, medullary thick ascending limb (mTAL) of loop of Henle and intercalated cells of the collecting tubule. In proximal tubular cells, CLC-5 was found to play an important role in receptor mediated endocytosis (RME). However, the functional roles of CLC-5 in mTAL and collecting tubules remain unclear. Since mTAL is normally exposed to a hypertonic environment, we aimed to examine the effect of hypertonicity on CLC5 expression in this nephron segment. Our studies revealed that exposure to hypertonicity (up to 550 mOsm) increased CLC-5 mRNA and protein levels in a murine mTAL cell line (MTAL), but not in an opossum kidney proximal tubular cell line (OK). A similar effect was also found in mouse kidneys, where CLC-5 expression was enhanced in renal medulla, but not cortex, after 48 hours of water deprivation. We also tested the effect of hypertonicity on endocytotic activity and found that exposure to hypertonicity caused a significant decrease in cellular uptake of FITC-labeled albumin in OK, but not in MTAL cells. Our results suggest that CLC-5 expression is upregulated by hypertonicity in medullary thick ascending limb cells but not in proximal tubular cells. We speculate that the increased CLC-5 levels in mTAL may serve to maintain the endocytotic activity in a hypertonic environment.