The three isoforms of both endothelin (ET) and big ET are not readily distinguishable by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) alone. We have previously used high-performance liquid chromatography (HPLC) linked with RIA to determine ET isoforms in various tissue extracts, and cell supernatants. Our aim was to confirm that the isoforms of ET secreted by human umbilical vein endothelial cells (HUVECs) are ET-1 and big ET-1, using HPLC linked to electrospray mass spectrometry (ESI-MS). Authentic synthetic peptide standards were used to obtain the specific ions for ET isoforms and to calculate their retention times on the HPLC system. ET-1, [Met7]-sulfoxy ET-1, ET-2, ET-3, and big ET-1 could all be separated by HPLC and by specific ions on ESI-MS. Pooled supernatant (500 ml) from 50 cultures of HUVECs was applied first to an anti-C-terminal ET-1 immunoaffinity column and then to an anti-C-terminal big ET-1 immunoaffinity column. The eluates were solid phase-extracted and then injected onto an HPLC system linked to ESI-MS. Peaks of mass intensity at the specific ions and retention times corresponding to ET-1, [Met7]-sulfoxy ET-1, and big ET-1 were found in these eluates. However, no signal was present for ET-2 or ET-3. These data confirm that HUVECs in culture produce ET-1, [Met7]-sulfoxy ET-1, and big ET-1.