The **data** collected from immunoprecipitation studies using mpkCCD 14 and HEK cells, combined with the results from GST pulldown assays strongly infers that AQP2 interacts with 14-3-3 and -. Furthermore, these selective 14-3-3 interactions being phosphorylation-dependent are supported by 1) an increased interaction of AQP2 with 14-3-3 following dDAVP treatment in a time scale where AQP2 phosphorylation is known to occur; 2) phosphatase inhibitors potentiating the dDAVP-induced AQP2/14-3-3 interactions; 3) a loss of AQP2 in GST pulldowns following -phosphatase treatment of prey lysates; 4) reduced AQP2/14-3-3 interactions in various AQP2 phosphorylation-deficient mutants; and 5) direct interaction of only Ser(P)-256 peptides with immobilized 14-3-3. Overlay assays and SPR analysis provided evidence that a direct 14-3-3/ AQP2 interaction was possible, which was critically dependent on Ser-256 phosphorylation. However, as 14-3-3 is known to associate with other proteins that can interact with AQP2, e.g. AKAP, Nedd4-2, Hsp70, BiP, ␤-actin, tropomyosin, PKA, and PP1 (46 -48), we cannot rule out that other proteins are involved in, or possibly stabilize the interaction, as part of a multiprotein complex. Interestingly, the interaction of AQP2 with 14-3-3 and -differs to other 14-3-3 membrane protein interactions observed in the kidney, e.g. the sodium hydrogen exchanger NHE1 association with 14-3-3␤ (49) and the UT-A urea transporter association with 14-3-3␥ (13), providing additional evidence that 14-3-3 isoforms are not redundant but rather play selective roles in modulating protein function.