Vasopressin triggers the phosphorylation and apical plasma membrane accumulation of aquaporin 2 (AQP2), and plays an essential role in urine concentration. Vasopressin, acting through protein kinase A, phosphorylates AQP2. However, the phosphorylation state of AQP2 could also be affected by the action of protein phosphatases (PPs). Rat inner medullas (IM) were incubated with calyculin (PP1 and PP2A inhibitor, 50 nM) or tacrolimus (PP2B inhibitor, 100 nM). Calyculin did not affect total AQP2 protein abundance (by Western blot) but did significantly increase the abundances of pS256-AQP2 and pS264-AQP2. It did not change pS261-AQP2 or pS269-AQP2. Calyculin significantly enhanced the membrane accumulation (by biotinylation) of total AQP2, pS256-AQP2, and pS264-AQP2. Likewise, immunohistochemistry showed an increase in the apical plasma membrane association of pS256-AQP2 and pS264-AQP2 in calyculin-treated rat IM. Tacrolimus also did not change total AQP2 abundance but significantly increased the abundances of pS261-AQP2 and pS264-AQP2. In contrast to calyculin, tacrolimus did not change the amount of total AQP2 in the plasma membrane (by biotinylation and immunohistochemistry). Tacrolimus did increase the expression of pS264-AQP2 in the apical plasma membrane (by immunohistochemistry). In conclusion, PP1/PP2A regulates the phosphorylation and apical plasma membrane accumulation of AQP2 differently than PP2B. Serine-264 of AQP2 is a phosphorylation site that is regulated by both PP1/PP2A and PP2B. This dual regulatory pathway may suggest a previously unappreciated role for multiple phosphatases in the regulation of urine concentration.