We used patch-clamp electrophysiology to investigate regulation of the epithelial Na⁺ channel (ENaC) by endothelin-1 (ET-1) in isolated, split-open rat collecting ducts. ET-1 significantly decreases ENaC open probability by ~3-fold within 5 minutes. ET-1 decreases ENaC activity through basolateral membrane ET_B but not ET_A receptors. In rat collecting duct, we find no role for phospholipase C (PLC) or protein kinase C (PKC) in the rapid response of ENaC to ET-1. ET-1, though, does activate src family tyrosine kinases and their downstream MAPK1/2 effector cascade in renal principal cells. Both src kinases and MAPK1/2 signaling are necessary for ET-1 dependent decreases in ENaC open probability in the split-open collecting duct. We conclude that ET-1 in a physiologically relevant manner rapidly suppresses ENaC activity in native, mammalian principal cells. These findings may provide a potential mechanism for the natriuresis observed in-vivo in response to ET-1, as well as, a potential cause for the salt-sensitive hypertension found in animals with impaired endothelin signaling.