We established a highly sensitive and specific sandwich-enzyme immunoassay (EIA) for endothelin-3 (ET-3), which showed no crossreactivity with endothelin-1 (ET-1), endothelin-2 (ET-2) and big-endothelin-1 (big-ET-1). We had previously established a sensitive sandwich-EIA for ET-1, which fully crossreacted with ET-2, but not with ET-3 or big-ET-1. These EIAs were used to examine the tissue distribution of immunoreactive (ir-) ET-3 and compare them with those of ir-ET-1 (including ir-ET-2) in Sprague-Dawley rats. High concentrations of ir-ET-3 were found in the intestine, lung, pituitary gland and brain (greater than 100 pg/g wet tissue), ir-ET-1(ET-2) showed widespread distribution, with large amounts in the lung and colon (greater than 1000 pg/g wet tissue). The pituitary gland was the only organ containing higher amounts of ir-ET-3 than ir-ET-1 (ET-2). In reverse phase-high performance liquid chromatography coupled with EIAs, the ir-ET-3 was exclusively eluted at the position of synthetic ET-3, indicating that the ir-ET-3 was identical to ET-3. The abundance of ET-3 in the intestine, pituitary gland and brain indicates that ET-3 is a new brain-gut peptide which may have a physiological function in nervous and endocrine systems.