Aquaporin-1 channels (AQP1) contribute to osmotically induced water transport in several organs including the kidney and serosal membranes such as the peritoneum and the pleura. In addition, AQP1 channels have been shown to conduct cationic currents upon stimulation by cyclic nucleotides. To date, the short-term regulation of AQP1 function by other major intracellular signalling pathways has not been studied. In the present study, we therefore investigated the regulation of AQP1 by protein kinase C. AQP1 wild type channels were expressed in Xenopus oocytes. Water permeability was assessed by hypotonic challenges. Activation of PKC by OAG induced a marked increase of AQP1-dependent water permeability. This regulation was abolished in mutated AQP1 channels lacking both consensus PKC phosphorylation sites T157 and T239 (termed AQP1 PKC). AQP1 cationic currents measured with double-electrode voltage-clamp were markedly increased after pharmacological activation of PKC by either OAG or PMA. Deletion of either T157 or T239 caused a marked attenuation of PKC-dependent current increases and deletion of both phosphorylation sites in AQP1 PKC channels abolished the effect. In vitro phosphorylation studies with synthesized peptides corresponding to amino acids 154-168 and 236-250 revealed that both T157 and T239 are phosphorylated by PKC. Upon stimulation by cyclic nucleotides, AQP1 wild type currents exhibited a strong activation. This regulation was not affected after deletion of PKC phosphorylation sites in AQP1 PKC channels. In conclusion, this is the first study to show that PKC positively regulates both water permeability and ionic conductance of AQP1 channels. This new pathway of AQP1 regulation is independent of the previously described cyclic nucleotide pathway and may contribute to the PKC stimulation of AQP1- modulated processes such as endothelial permeability, angiogenesis and urine concentration.