The epithelial sodium channel (ENaC) in distal nephron plays an important role in regulating sodium balance to control systemic blood pressure and consequently affect cardiovascular function. Using patch-clamp techniques, we found that application of ionomycin (a Ca2+ ionophore) to A6 cells reduced open probability of ENaC from 0.56±0.17 to 0.09±0.05 (initial inhibition; p<0.01) and 0.07±0.06 (sustained inhibition; p<0.01) in 5 cell-attached patches. GF 109203X, a protein kinase C (PKC) inhibitor, did not affect the initial inhibition, but abolished the sustained inhibition. In contrast, U-73122, a phospholipase C (PLC) inhibitor, but not U-73343 (an inactive analog of U-73122), abolished the initial inhibition and partially reduced the sustained inhibition. Stimulation of PKC with phorbol 12-myristate 13 acetate (PMA) inhibited ENaC with a 3-min delay in control cells, but not in the cells pretreated with U-73122. Using confocal microscopy, we found that ionomycin induced a significant decrease in membrane phosphatidylinositol-4,5-bisphosphate (PIP2), monitored by a GFP-tagged pleckstrin homology domain of PLC-d1. Like ENaC activity, the sustained decrease in membrane PIP2 was abolished by inhibition of PKC, while the initial decrease in membrane PIP2 induced by ionomycin was abolished by inhibition of PLC. Our results suggest that the secondary messenger Ca2+ alters ENaC activity through both PLC- and PKC-dependent decreases in membrane PIP2.