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 the role of sphingolipid- and cholesterol-rich microdomains, called lipid rafts, in ENaC regulation. Methyl-b- cyclodextrin (MBCD) is a widely used tool to deplete cholesterol from the plasma membrane thereby destroying lipid rafts. We investigated the effect of MBCD on ENaC function in X. laevis oocytes heterologously expressing ratENaC. To confirm the MBCD effect in the oocyte plasma membrane, we used fluorescent staining of cholesterol with filipin and of ganglioside GM-1, a lipid raft marker, with choleratoxin subunit B. Live cell staining demonstrated that treatment with MBCD largely reduced filipin and choleratoxin subunit B staining over time. This indicates that the integrity of lipid rafts was severely impaired by the MBCD treatment. Interestingly, baseline whole-cell ENaC currents were only slightly reduced by 20 mM MBCD. In contrast, in outside-out patch clamp recordings the known stimulatory effect of recombinant SGK1 in the pipette solution (Diakov & Korbmacher 2006 J Biol Chem 279: 38134) was essentially abolished in oocytes pretreated with MBCD. These results indicate that ENaC activation by SGK requires presence of intact lipid rafts which are thought to serve as signalling platforms.