Aquaporin-2 (AQP2) is a water channel in collecting duct principal cells in the kidney. Vasopressin catalyzes AQP2 phosphorylation at several serine sites in its C-terminus: Ser-256, Ser-261, and Ser-269. Upon stimulation by vasopressin, Ser-269 phosphorylation increases and Ser-261 phosphorylation decreases. Ser-256 phosphorylation is relatively constant. However, whether these types of phosphoregulation occur independently in distinct AQP2 populations or sequentially in the same AQP2 population is unclear. Especially, the manner of vasopressin-mediated Ser-261 phospho-regulation has been in controversy. In this study, we established phospho-specific AQP2 immunoprecipitation assays and investigated how pS256-positive AQP2 and pS269-positive AQP2 are catalyzed by forskolin or vasopressin, focusing on their Ser-261 phosphorylation status in polarized Madin-Darby canine kidney (MDCK) cells and in mice. In forskolin-treated MDCK cells, Ser-269 phosphorylation preceded Ser-261 dephosphorylation and Ser-256 phosphorylation was constant. In both MDCK cells and mouse kidney, phospho-specific immunoprecipitation revealed that the regulated Ser-269 phosphorylation occurred in the pS256-positive AQP2 population. Importantly, basal-state Ser-261 phosphorylation and its regulated dephosphorylation occurred in the pS256-and pS269-positive AQP2 population. These results provide the direct evidence that the Ser-261 dephosphorylation is involved in the pS256- and pS269-related AQP2 regulation. (C) 2016 Elsevier Inc. All rights reserved.