Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP2 with anti-PIP2 antibody, hydrolysis of PIP2 with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-L-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP2 and PIP3 significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat a-, β-, and γ-ENaC. However, PIP2 and PIP3 did not affect surface expression of ENaC, indicating that PIP2 and PIP3 regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors.