Activity of the epithelial sodium channel (ENaC) is limiting for Na⁺ reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) modulate the activity of this channel. Activation of purinergic receptors triggers multiple events including activation of protein kinase C (PKC) and phospholipase C (PLC) with the latter depleting plasma membrane PI(4,5)P₂. Here, we investigate regulation of ENaC in renal principal cells by purinergic receptors via PLC and PI(4,5)P₂. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)P₂ levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor, U73122, but not its inactive analogue, U73343, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting either purinergic receptors or PLC in resting cells dramatically increased ENaC activity above basal levels indicating tonic activation of purinergic signaling in these polarized renal epithelial cells. Increased ENaC activity was associated with elevation of in apical membrane PI(4,5)P₂ levels. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)P₂ levels. Dwell time analysis reveals that depletion of PI(4,5)P₂ forces ENaC toward a closed state. In contrast, increasing PI(4,5)P₂ levels above basal values locks the channel in an open state interrupted by brief closings. Thus, our results suggest that purinergic control of apical membrane PI(4,5)P₂ levels is a major regulator of ENaC activity in renal epithelial cells.