Activation of basolateral V₂ receptors by vasopressin increases water reabsorption in the collecting duct (CD) via a series of intracellular events that culminate in increased insertion of aquaporin-2 water channels (AQP2) into the apical membrane. Recent studies have shown that this process is inhibited by extracellular ATP acting on P2 receptors expressed on the basolateral membrane of CD principal cells, and that P2Y₂-like receptors are responsible (Unwin et al. 2003). The present study has used the Xenopus oocyte expression system to investigate whether other P2 receptor subtypes can influence AQP2 function, and has localised P2 receptor distribution in the rat CD. Defolliculated Xenopus oocytes (co-expressing AQP2 and a given P2 receptor) were placed in a hypotonic (10 mosm/l) Barth’s solution, and AQP2-mediated swelling was monitored by video imaging at 1.7 s intervals for 1 min; from this, osmotic water permeability (P_f) was calculated (Deen et al. 1994). When P2X₂, P2Y₂ or P2Y₄ receptors were co-expressed with AQP2, then activated by 10 µM ATP, P_f was reduced significantly (by 46 ± 8% (P<0.01, unpaired t test), 53 ± 7% (P<0.01) and 57 ± 3% (P<0.01), respectively; means ± SEM; n=8 in each case). Western blot analysis, with a specific AQP2 antibody, confirmed that the P2 receptor-mediated inhibition of cell swelling was due to removal of AQP2 protein from the plasma membrane in each case (n=3). Kidneys from three terminally anaesthetised adult Sprague Dawley rats were perfusion-fixed with paraformaldehyde (4%) and sliced (8 µm). Using antibodies specific for P2X₂, P2Y₂ and P2Y₄ receptors, immunohistochemical analysis demonstrated the expression of P2X₂ and P2Y₄ receptors on the apical membrane throughout the CD and P2Y₂ on the basolateral membrane in the inner medullary CD. These results show directly that activation of certain P2 receptors can inhibit AQP2-mediated water transport. They also suggest that, in addition to the previously documented effect of basolateral P2Y₂ receptors, apically located P2 receptors (P2X₂ or P2Y₄) might play a role in modulating vasopressin-stimulated water reabsorption in the CD, thus strengthening suggestions that intraluminal nucleotides might act as paracrine/autocrine agents.