Endothelins regulate nephron sodium and water transport, prostaglandin E2 (PGE2) synthesis, and phospholipid metabolism. Recent studies suggest that renal tubule cells synthesize endothelins. To determine which nephron sites have such potential, endothelin production by cells derived from different nephron segments was examined. Immunoreactive endothelin 1 (ET-1) and endothelin 3 (ET-3) were measured in supernatants of cultured rabbit proximal tubule (PT), medullary thick ascending limb (MTAL), cortical collecting tubule (CCT), and inner medullary collecting duct (IMCD) cells. All cell types released immunoreactive ET-1 and ET-3. However, the amounts of endothelin produced differed as follows: IMCD greater than MTAL greater than CCT much greater than PT for ET-1 and IMCD greater than MTAL = PT = CCT for ET-3; in all cases ET-1 much greater than ET-3. To confirm de novo ET-3 synthesis, IMCD cells were labeled with [35S]cysteine, and the supernatant was immunoprecipitated with anti-ET-3 antibody. Sample and standard ET-3 eluted at identical positions on high-performance liquid chromatographs, confirming de novo synthesis of ET-3 by cultured IMCD cells. These data raise the possibility of an important functional role for nephron-derived endothelin and, in particular, endothelin produced by tubule cells in the medulla.