Increases in intracellular calcium (Ca(2+)) inhibit renal sodium (Na(+)) absorption in cortical collecting ducts, but the precise mechanism is unclear. We, therefore, studied the effects of raising intracellular Ca(2+) (using 10 µmol/L A23187, a Ca(2+) ionophore) on wild-type and Liddle-mutated human epithelial Na(+) channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. A23187 decreased amiloride-sensitive Na(+) current by 55% in oocytes expressing wild-type hENaC, an effect prevented by co-exposure to 50 μmol/L W-7 (to inhibit the Ca(2+)/calmodulin complex). By contrast, co-exposure to 50 μmol/L calphostin (to inhibit protein kinase C) or 5 μmol/L KN-62 (to inhibit Ca(2+)/calmodulin-dependent protein kinase II) had no effect on the decrease in amiloride-sensitive Na(+) current elicited by A23187 alone. Whereas A23187 reduced amiloride-sensitive Na(+) current in oocytes expressing wild-type hENaC, it had no similar effect in those expressing Liddle-mutated hENaCs, suggesting that the activity of individual Na(+) channels in situ was unchanged by the rise in intracellular Ca(2+). These data suggest that the A23187-induced rise in intracellular Ca(2+) inhibited wild-type hENaC through a W-7-sensitive mechanism, which likely reflected enhanced removal of Na(+) channels from the cell membrane by endocytosis. We, therefore, propose that Na(+) absorption in cortical collecting duct cells is inhibited by Ca(2+), possibly when complexed with calmodulin.