The epithelial Na⁺ channel (ENaC) plays a major role in the regulation of Na⁺ balance and blood pressure by controlling Na⁺ reabsorption along the renal collecting duct (CD). ENaC activity is inhibited by P2 receptors (P2Rs) which are activated by extracellular nucleotides. However, uncertainties exist over the mechanism of inhibition, the P2R subtype(s) involved and molecular pathway(s) activated. This study has addressed the relationship between apical P2Rs and ENaC inhibition in the rat CD. Kidneys from terminally anaesthetized adult Sprague-Dawley rats, which had been maintained on a low (0.01%) Na⁺ diet (for 10-days in order to increase CD ENaC expression), were microdissected and isolated CD segments were split-open to expose the apical membrane of individual principal cells (PCs). PCs (in situ) were studied under voltage-clamp conditions (V_h = -60 mV) using the whole-cell perforated-patch clamp technique. Apically-applied ATP, ATPγS, UTP and 2meSATP each evoked inward currents (≤500 pA; 10 µM; n = 10), whereas 2meSADP and BzATP were inactive (10 μM; n = 10). Currents evoked by 2meSATP were insensitive to DIDS (100 µM; n = 5) and shown to be cationic, consistent with P2X channel activation. Currents evoked by UTP were abolished by DIDS (n = 5), consistent with P2Y receptor activation of Ca²⁺-dependent Cl^– currents. Within 3 minutes, nucleotide evoked inward currents significantly reduced the amplitude of subsequent ENaC-mediated currents over a wide range of holding voltages (between 30-60%; P<0.05) without changing the inward rectification or reversal potential (n = 12). Lowering extracellular Na⁺ concentration from 145 to 50 mM failed to alter the degree of inhibition caused by UTP-evoked currents, however 2meSATP-evoked responses instead potentiated the amplitude of the ENaC-mediated current-voltage relationships (75%; n = 7). 2meSATP-mediated potentiation of ENaC currents was abolished by wortmannin (100 nM; n = 3). When taken together with previously reported immunohistochemical data (Chapman et al. 2006), these biophysical and pharmacological data suggest that ENaC activity is inhibited by apical P2Y_{2 and/or 4} and P2X_{4 and/or 4/6} receptors when extracellular Na⁺ concentrations are high (145 mM; n = 12) but apical P2X_{4 and/or 4/6} receptors switch to potentiators of ENaC activity (involving PI3K) when extracellular Na⁺ is lowered (to 50 mM; n = 5). We propose that P2X_{4 and/or 4/6} receptors function as apical Na⁺ sensors responsible for local regulation of ENaC activity in the CD and could thereby regulate Na⁺ balance and systemic blood pressure.