Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of CD Na+ reabsorption. Salt loading is thought to increase CD ET-1 production; however, definitive evidence of this, as well as understanding of the mechanisms transducing this effect, is lacking. Tubule fluid flow increases in response to Na+ loading; hence, we studied flow modulation of CD ET-1 production. Three days of a high-salt diet increased mouse and rat inner medullary CD (IMCD) ET-1 mRNA expression. Acute furosemide infusion increased urinary ET-1 excretion in anesthetized rats. Primary cultures of mouse or rat IMCD detached in response to flow using a closed perfusion chamber, consequently a CD cell line (mpkCCDcl4) was examined. Flow increased ET-1 mRNA at shear stress rates exceeding 1 dyne/cm2, with the maximal effect seen between 2 and 10 dyne/cm2. Induction of ET-1 mRNA was first evident after 1 h, and most apparent after 2 h, of flow. Inhibition of calmodulin or dihydropyridine-sensitive Ca2+ channels did not alter the flow response; however, chelation of intracellular Ca2+ or removal of extracellular Ca2+ largely prevented flow-stimulated ET-1 mRNA accumulation. Downregulation of protein kinase C (PKC) using phorbol 12-myristate 13-acetate, or PKC inhibition with cal-phostin C, markedly reduced flow-stimulated ET-1 mRNA levels. Flow-stimulated ET-1 mRNA accumulation was abolished by inhibition of phospholipase C (PLC). Taken together, these data indicate that flow increases CD ET-1 production and this is dependent on extracellular and intracellular Ca2+, PKC, and PLC. These studies suggest a novel pathway for coupling alterations in extracellular fluid volume to CD ET-1 production and ultimately control of CD Na+ reabsorption. © 2011 the American F650 Physiological Society.