Vasopressin (VP) regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the C-terminus, and among them, serine-256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of the serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells, and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and western blotting with phospho-specific antibodies. Using methyl-b-cyclodextrin (MBCD), cold block or bafilomycin, and Taxol, we blocked AQP2 at the plasma membrane, in the peri-nuclear trans-Golgi network (TGN), and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared to baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the TGN, or in cytoplasmic vesicles, and that this event is dependent on the expression of PKA in these cells.