Molecular forms and concentrations of endothelin (ET) family peptides in normal human plasma and urine were investigated using five different sandwich-type enzyme immunoassays (sandwich-EIAs) and reverse-phase high performance liquid chromatography (RP-HPLC). We found that immunoreactive (IR-) big ET-3 and IR-big ET-2 were the major species in both plasma and urine from normal humans. Big ET-2(1-38) was found to be the major form of IR-big ET-2 in plasma. Urinary IR-big ET-2 and plasma and urinary IR-big ET-3 were heterogeneous, and the IR-big ET-3s were larger (M(r) 6,000-12,000) than authentic big ET-3. IR-big ET-1, IR-ET-1, and IR-ET-3 were found at relatively low concentrations in human plasma and urine, and their major forms were identical to authentic big ET-1, ET-1, and ET-3, respectively. Although ET-1 and other ET family peptides have often been measured using RIAs with board-spectrum antibodies, the presence of high concentrations of IR-big ET-2 and IR-big ET-3, but not IR-ET-1, in plasma and urine indicates that the clinical significance of ET family peptides should be investigated with assay methods specific to each ET and big ET.