--- - |2+ Key Points Aquaporin‐2 (AQP2) is crucial for water homeostasis, and vasopressin (VP) induces AQP2 membrane trafficking by increasing intracellular cAMP, activating PKA, and causing phosphorylation of AQP2 at serine 256, 264 and 269 residues and dephosphorylation of serine 261 residue on the AQP2 c‐terminus. It is thought that serine 256 is the master regulator of AQP2 trafficking, and its phosphorylation has to precede the change of phosphorylation state of other serine residues. We found that Src inhibition causes serine 256 independent AQP2 membrane trafficking and induces phosphorylation of serine 269 independent of serine 256. This targeted phosphorylation of serine 269 is important for Src inhibition induce AQP2 membrane accumulation. Without serine 269, Src inhibition exerts no effect on AQP2 trafficking. This result helps us better understand the independent pathways that can target different AQP2 residues, and design new strategies to induce or sustain AQP2 membrane expression when VP signaling is defective. Abstract Aquaporin‐2 (AQP2) is essential for water homeostasis. Upon stimulation by vasopressin, AQP2 is phosphorylated at serine 256 (S256), S264 and S269, and dephosphorylated at S261. It is thought that S256 is the master regulator of AQP2 trafficking and membrane accumulation, and that its phosphorylation has to precede phosphorylation of other serine residues. In this study, we found that VP reduces Src kinase phosphorylation: by suppressing Src using the inhibitor dasatinib and siRNA, we could increase AQP2 membrane accumulation in cultured AQP2‐expressing cells and in kidney collecting duct principal cells. Src inhibition increased exocytosis and inhibited clathrin‐mediated endocytosis of AQP2, but exerted its effect in a cAMP, PKA and S256 phosphorylation (pS256) independent manner. Despite the lack of S256 phosphorylation, dasatinib increased phosphorylation of S269, even in S256A mutant cells in which S256 phosphorylation cannot occur. To confirm the importance of pS269 in AQP2 re‐distribution, we expressed an AQP2 S269A mutant in LLC‐PK1 cells, and found that dasatinib no longer induced AQP2 membrane accumulation. In conclusion, Src inhibition causes phosphorylation of S269 independently of pS256, and induces AQP2 membrane accumulation by inhibiting clathrin‐mediated endocytosis and increasing exocytosis. We conclude that S269 can be phosphorylated without pS256, and pS269 alone is important for AQP2 apical membrane accumulation under some conditions. These data increase our understanding of the independent pathways that can phosphorylate different residues in the AQP2 c‐terminus, and suggest new strategies to target distinct AQP2 serine residues to induce membrane expression of this water channel when VP signaling is defective. This article is protected by copyright. All rights reserved - 'The Journal of Physiology, Volume 0, Issue ja, -Not available-. '