Epithelial Na⁺ channels (ENaC) play a crucial role in Na⁺ transport in the distal nephron, where they co-localize with ATP-gated P2 receptors. Evidence suggests that ATP may indirectly regulate ENaC by first activating luminal P2 receptors (Unwin et al. 2003). Previously, using in vivo microperfusion of the rat collecting duct (CD), ²²Na⁺ urinary recovery was measured to assess Na+ reabsorption following P2 receptor activation. Although the non-hydrolysable ATP analogue ATPγS had no effect in control animals, ATPγS inhibited Na+ reabsorption in Na⁺-restricted rats (Shirley et al. 2001). Other non-hydrolysable analogues (2meSADP, BzATP and Ap₄A) had no measurable effect on ²²Na⁺ uptake (unpublished observation). In the present study, we have investigated the selectivity and potency of this series of ATP analogues on recombinant ionotropic P2X and metabotropic P2Y receptors found in rat CD, expressed in Xenopus oocytes and studied under voltage-clamp conditions, in order to compare in vitro agonist profiles with our in vivo findings in Na⁺-restricted rats. Immunohistochemical and RT-PCR studies have identified P2Y₂ and P2Y₄ receptors, homomeric P2X₄, P2X₅ and P2X₆ receptors, and the possibility of heteromeric P2X_4/5, P2X_4/6 and P2X_5/6 receptors in rat CD (Turner et al. 2003). For all the above recombinant P2 receptors, agonist concentration-response curves (100nM-1mM) were constructed. ATPγS was found to be a full agonist equipotent with ATP at all receptors tested. BzATP was found to be a partial agonist at P2X₅ and P2X_4/5. Ap₄A was a full agonist at P2Y₂ and P2Y₄ receptors and a partial agonist equipotent with ATP at P2X₄ and P2X₅. 2meSADP (1mM) was inactive at all the receptors tested. In conclusion, given the agonist activity of BzATP and Ap₄A in vitro, and assuming a lack of function of homomeric P2X₆, it is unlikely that P2Y₂, P2Y₄, P2X₄, P2X₅, P2X₆ and P2X_4/5 receptors play a role in the inhibition of Na⁺ reabsorption in sodium-restricted rats. In contrast, a role may exist for the heteromeric P2X_4/6 and/or P2X_5/6 receptors. Interestingly, Na⁺ and Ca²⁺ influx through the P2X_4/6 (not P2X_5/6) ion channel, following its activation by extracellular ATP, results in the down-regulation of ENaC (Wildman et al. 2003). Our findings suggest a potential regulatory role for P2X_4/6 in Na⁺-restricted rats, where increased expression of ENaC in the distal nephron would be expected.