Aquaporin-4 (AQP4) plays an important role in the basolateral movement of water in the collecting duct. Here we show that this water channel can be dynamically regulated. Water permeability (P _f) was measured in individual LLC-PK₁ cells that were transiently transfected with AQP4. To identify which cells were transfected, AQP4 was tagged at the NH₂ terminus with green fluorescent protein. Transfected cells showed a strong fluorescent signal in basolateral membrane and a low-to-negligible signal in the cytosol and apical membrane. Activation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) significantly decreasedP _f of cells expressing AQP4 but had no effect on neighboring untransfected cells. No redistribution of AQP4 in response to PDBu was detected. Dopamine also decreased theP _f in transfected cells. The effect was abolished by the PKC inhibitor Ro 31–8220. Reduction of AQP4 water permeability by PDBu and dopamine was abolished by point mutation of Ser¹⁸⁰, a consensus site for PKC phosphorylation. We conclude that PKC and dopamine decrease AQP4 water permeability via phosphorylation at Ser¹⁸⁰ and that the effect is likely mediated by gating of the channel.