The transformed human endothelial cell line EA.hy926 is commonly used for studying in vitro different aspects of endothelial cell biology such as signal transduction, expression or angiogenesis. These cells have the ability to process big endothelin (big-ET) into endothelin (ET), and express the endothelin-converting enzyme ECE-1. Several isoforms of ECE-1 which differ only in their N-terminal part (i.e. the end of the cytosolic domain) have now been identified. We could detect the co-expression of all four isoforms. Recent works have shown that the variable cytosolic domain is responsible for the differential intracellular localization of ECE-1 isoforms. Using antibodies directed against ECE-1a and ECE-1b/c/d, we have characterized the intracellular distribution of these isoforms in EA.hy926 cells by immunofluorescence. Electron microscopy allowed us to identify further the intracellular compartment that contains ECE-1 as multivesicular bodies, a compartment involved in the endocytic pathway. In addition, using an antibody directed against the catalytic domain, we could demonstrate that no monomeric ECE-1 is present at the plasma membrane. Indeed, detection of ECE-1 immunoreactivity at the cell surface of living cells required a dithiothreitol (DTT) treatment. Altogether, these results demonstrate that the EA.hy926 cell line is a helpful model for studying the regulation of the production of endothelin by ECE.