Aquaporin-5 (AQP5) is a water channel playing crucial role for exocrine function. Since the water pore of AQPs are always opened, their functions are thought to be regulated by translocalization between plasma membrane and intracellular space. The regulation of translocalization of AQP2 in collecting ducts was well investigated, and the importance of its C-terminal domain has been established. In the present study, therefore, we examined the effects of various AQP5 C-terminal deletions or mutations on cell surface expression. Subcellular localization of AQP5 mutants was observed by immunofluorescence assay. AQP5 mutant deleting C-terminal 10 amino acids (²⁵⁶RKKTMELTTR²⁶⁵) located only in cytosol, while full-length AQP5 was mainly on plasma membrane. Deleting some more amino acids did not recover this abnormal localization. AQP5 mutants in cytosol did not co-localize with EEA1 and endocytosis inhibitor did not affect their localization, suggesting their abnormal localization is not due to increased endocytosis. Additionally, AQP5 mutant with substitutions of Ala for Met²⁶⁰, Glu²⁶¹ or Leu²⁶² showed similar abnormal localization. Therefore, we assume that these three amino acids in AQP5 are required for its localization at plasma membrane, and that there may be novel mechanism is due to neither phosphorylation nor ubiquitination.