Antidiuretic hormone modulates the water permeability (P(f)) of epithelial cells in the rat kidney by vesicle-mediated insertion and removal of the aquaporin-2 (AQP-2) water channel. AQP-2 possesses a single consensus cAMP- dependent protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to regulate channel P(f) (Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F. (1995) J. Biol. Chem. 270, 1038410387). To test whether PKA phosphorylation of AQP-2 alters channel P(f), we compared the P(f) values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [γ-32P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove 32P label from AQP-2. However, the P(f) (0.16 ± 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 ± 0.3 mol of PO4/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 ± 8% of the phosphate has been removed (P(f) 0.14 ± 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2's P(f). Instead, AQP-2 phosphorylation by PKA may modulate AQP-2's distribution between plasma membrane and intracellular vesicle compartments.