Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na⁺ channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P₃ and PI(4,5)P₂ to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in β- and γ- but not α-ENaC as necessary for PI(3,4,5)P₂ but not PI(4,5)P₂ modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in β- and γ-ENaC are critical to PI(3,4,5)P₃ augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of β- and γ-ENaC were identified as being critical to down-regulation of ENaC activity and P_o in response to depletion of membrane PI(4,5)P₂. These regions of the channel played no identifiable role in a PI(3,4,5)P₃ response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P₂ to increase open probability. We conclude that β and γ subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P₃ and PI(4,5)P₂. This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.