The amiloride-sensitive epithelial Na+ channel (ENaC) is usually found in the apical membrane of epithelial cells, but is also described in endothelial cells. Since ENaC is thoroughly examined in epithelial cells little is known about the Na+ channel in the endothelium. Therefore, we studied the influence of aldosterone (aldo), spironolactone (spiro) and amiloride on the abundance of ENaC in the plasma membrane (PM) of human endothelial cells (HUVEC) and in intracellular stores. With quantum-dot labeled antibodies we detected and quantified ENaC molecules in the PM of HUVEC. For the determination of the total amount of ENaC in the cells Western blots were made and apical cell surfaces were scanned with AFM. We found that aldo increases the quantity of ENaC located in the PM and also the total amount of ENaC per cell. In addition, aldo treatment enhances the apical surface area of HUVEC. Amiloride application of aldo-pretreated cells diminished ENaC by approximately 87%. At the same time, the apical surface area was found dramatically decreased. Similarly, spiro reduced the abundance of ENaC in membrane and cell. We conclude that in endothelial cells aldo induces ENaC expression and insertion into the PM. By blocking ENaC with amiloride the channel disappears from the cell surface and from intracellular pools indicating either rapid degradation or membrane pinch-off.