Whole body water homeostasis is regulated by signaling cascades activated following stimulation of a range of 7-transmembrane receptors in the kidney. These receptors signal through heterotrimeric G proteins, and include the type 2 vasopressin receptor (V2R) and prostaglandin receptors EP2 and EP4.The G protein α-subunit mediates cAMP signaling, which is thought to play a major role in increasing water transport by increasing aquaporin-2 (AQP2) apical membrane accumulation in the collecting duct. The role of other G protein subunits, namely of βγ subunits, for AQP2 regulation is unknown and is the focus of this study. AQP2 apical membrane accumulation was investigated by cell surface biotinylation in AQP2-transfected Madin Darby Canine Kidney (MDCK) cells and in a mouse cortical collecting duct cell line (mpkCCD cells) after 10 min preincubation with either vehicle, the Gβγ inhibitor gallein (100 µM) and/or the adenylyl cyclase inhibitor SQ22536 (500 µM) followed by stimulation with agonists for EP2 (butaprost 50 nM), EP4 (CAY10598 1.3 µM) or V2R (dDAVP 1 nM). In MDCK cells, gallein abolished AQP2 apical membrane accumulation induced by CAY10598, but had no significant effect on butaprost induced AQP2 apical membrane accumulation. SQ22536 prevented butaprost induced cAMP increases, but not AQP2 apical membrane accumulation. When co-administered, SQ22536 and gallein prevented butaprost induced apical membrane accumulation of AQP2. In mpkCCD cells, SQ22536 had no effect on dDAVP induced AQP2 apical membrane accumulation, but when co-administered, SQ22536 and gallein reduced dDAVP-induced AQP2 apical membrane accumulation compared with preincubation with SQ22536 alone. Preliminary data indicate that mice subjected to 18h water restriction after treatment with gallein (30 mg/kg) had lower urine osmolality compared with vehicle-treated mice. In conclusion, the Gβγ subunit is involved in AQP2 apical membrane accumulation in epithelial cell lines and may partially determine urinary concentrating mechanism in vivo. This signaling pathway occurs in parallel to the cAMP signaling cascade during stimulation of the EP2 receptor and of the V2R, but is the only EP4 mediated pathway involved in AQP2 membrane accumulation.