In the renal collecting duct (CD), water reabsorption is determined by the expression of aquaporin-2 (AQP2) in the apical membrane of principal cells, which in turn is dependent on the activation of vasopressin receptors in the basaolateral membrane. This process is inhibited by extracellular ATP, which acts on P2 receptors expressed on the basolateral membrane of CD principal cells, and there is evidence that P2Y₂-like receptors are responsible (Unwin et al. 2003). The present study has used immortalized mouse mpkCCD(cl4) CD principal cells to investigate whether other P2 receptor subtypes may influence vasopressin-induced AQP2 expression. Cultured mpkCCD(cl4) cells were grown to confluence on transwell permeable supports, then treated with 1 nM dDAVP (applied to the basal media) for 96 h to induce AQP2 expression. In some experiments, cells were also treated with 1 µM ATPγS (applied to the apical media) for the final 2 h prior to termination of dDAVP treatment. Confluent monolayers were then washed in phosphate-buffered saline and fixed with paraformaldehyde (4% for 10 min) before performing immunofluorescence labelling (using P2- and AQP2-specific antibodies) and confocal microscopy to investigate AQP2 and P2 receptor expression patterns. In monolayers of cells untreated with dDAVP (n=5), immunostaining for AQP2 was not evident. In contrast, positive staining for P2Y₁ and P2Y₄ receptors was seen in the apical region, whereas P2X₂, P2X₄, P2X₅, P2X₆ and P2X₇ receptor subunits and P2Y₂, P2Y₁₁ and P2Y₁₂ receptors were located throughout the cytoplasm up to the cell borders and more strongly in perinuclear regions. Treatment with dDAVP (n=5) resulted in positive immunostaining for AQP2, P2X₁ and P2X₂ in the apical region and basolateral staining for P2Y₂. Staining for all other P2 receptors remained unchanged. In monolayers treated with dDAVP and ATPγS (n=3), AQP2 and P2X₁ staining in the apical region was weak and vesicular, whereas all other P2 receptor staining remained unchanged. These results suggest that dDAVP may regulate the expression of some P2 receptors (P2X₁ apically, P2X₂ apically and P2Y₂ basolaterally), as well as AQP2. They also suggest that apically located P2 receptors (P2Y₁, P2Y₄, P2X₁ and/or P2X₂) might play a role in modulating vasopressin-stimulated water reabsorption in the CD by inhibiting the expression or trafficking of AQP2. These findings thus reinforce the suggestion that intraluminal nucleotides might act as paracrine/autocrine agents in the nephron.