Recent evidence suggests that the lipid environment of the epithelial sodium channel (ENaC) may be relevant for its function. The aim of this study was to provide functional evidence for ENaCs association with sphingolipid-and cholesterol-rich microdomains, called lipid rafts. Methyl-b-cyclodextrin (MBCD) is a widely used tool to deplete cholesterol from the plasma membrane thereby destroying lipid rafts. We tested the effect of MBCD on the amiloride-sensitive short circuit current (I_SC-Ami ) of M-1 mouse renal collecting duct cells and on the amiloride-sensitive whole-cell currents ([Delta]I_Ami ) of Xenopus laevis oocytes heterologously expressing ratENaC. Both, I_SC-Ami and [Delta]I_Ami were significantly reduced by MBCD. Detergent solubilization followed by sucrose density gradient centrifugation confirmed that MBCD effectively destroyed lipid rafts since the lipid raft marker caveolin-1 was shifted to a higher density fraction. Interestingly, treating M-1 cells with forskolin to stimulate the channel by an increase in intracellular cAMP shifted ENaC but not Nedd4 or caveolin-1 out of the lipid raft fraction. We conclude that intact lipid rafts are required for normal ENaC function. The forskolin induced redistribution of ENaC away from the lipid rafts may contribute to ENaC stimulation by cAMP by dissociating the channel from its inhibitory protein Nedd4.