The potent vasoconstrictor endothelin-1 (ET-1) is secreted constitutively by endothelial cells and has been implicated in the pathophysiology of several cardiovascular diseases. It is generated from its inactive intermediate, big ET-1, through the action of endothelin-converting enzyme (ECE). Using several complementary techniques, we have demonstrated that ECE is present at the cell surface and on intracellular vesicles and that it recycles from the cell surface in endothelial cells. This is the first ultrastructural localization of ECE in lung and the first time big ET-1 and ECE have been colocalized by immunogold in a vesicular population, 50 to 100 nm in diameter. In addition, by double immunogold staining of ultrathin cryosections, we have localized ECE together with angiotensin-converting enzyme on the luminal membrane of endothelial cells. With cell surface biotinylation of a transformed rat endothelial cell line and of human umbilical vein endothelial cells, we have confirmed the presence of ECE on the plasma membrane. After treatment of endothelial cells with chloroquine, ECE and trans-Golgi network 38 protein were shown by immunofluorescence staining to localize to the same intracellular compartment.