The polarized nature of epithelial cells allows for different responses to luminal or serosal stimuli. In kidney tubules, ATP is produced luminally in response to changes in luminal flow. Luminal increases in ATP have been previously shown to inhibit the renal epithelial Na⁺ channel (ENaC). On the other hand, ATP is increased basolaterally in renal epithelia in response to aldosterone. We tested the hypothesis that basolateral ATP can stimulate ENaC function through activation of the P2X₄ receptor/channel. Using single channel cell-attached patch-clamp techniques, we demonstrated the existence of a basolaterally expressed channel stimulated by the P2X₄ agonist 2-methylthio-ATP (meSATP) in Xenopus A6 cells, a renal collecting duct principal cell line. This channel had a similar reversal potential and conductance to that of P2X₄ channels. Cell surface biotinylation of the basolateral side of these cells confirmed the basolateral presence of the P2X₄ receptor. Basolateral addition of meSATP enhanced the activity of ENaC in single channel patch-clamp experiments, an effect that was absent in cells transfected with a dominant negative P2X₄ receptor construct, indicating that activation of P2X₄ channels stimulates ENaC activity in these cells. The effect of meSATP on ENaC activity was reduced after chelation of basolateral Ca²⁺ with EGTA or inhibition of phosphatidylinositol 3-kinase with LY-294002. Overall, our results show that ENaC is stimulated by P2X₄ receptor activation and that the stimulation is dependent on increases in intracellular Ca²⁺ and phosphatidylinositol 3-kinase activation.