A surface plasmon resonance-based biosensor (BIA technology) and enzyme-linked immunosorbent assays (ELISA) have been used for detecting and characterizing human endothelin (ET), a potent vasoactive 21 amino acid polypeptide. Antibodies produced against the isoform ET-1 and its C-terminal eptapeptide ET-1 15–21 have been characterized with respect to their binding capacity to the two isoforms ET-1 and ET-3, the non-secreted portion of the precursor molecule Big.ET-1 22–38, the C-terminal of ET-1, six analogues of ET-1 16–21 each containing a substitution with Ala of a single amino acid in positions 16–21, respectively, and three synthetic cyclic peptides mimicking the N-terminal portion of ET-1. Antibodies reacting with ET-1 also bound to ET-1 16–21 but did not cross-react with ET-3 or Big.ET-1 22–38. Ala substitution in positions 16, 17 and 19 of ET-1 16–21 hardly affected the antibody binding capacity of ET-1 16–21, whereas Ala substitution of Asp 18, Ile 20 and, in particular, Trp 21, inhibited its immunoreactivity. The C-terminus thus represents an immunodominant epitope in ET-1 and is important for antibody binding. Epitope mapping using as antibody pairs polyclonal anti-ET-1 and monoclonal anti-ET-1 15–21 antibodies indicated the presence of another immunogenic domain in the N-terminal portion of the molecule. There was excellent agreement between the epitopes determined using ELISA and BIA analyses.