The purpose of this study was to investigate the effects of inhibitors of the Na⁺, K⁺-pump and membrane depolarizing agents on endothelium-dependent acetylcholine-induced relaxation in the rabbit thoracic aorta. Aortic rings were prepared from the rabbit descending thoracic aorta and the contractility of the ring was measured in various conditions such as application of ouabain, exposure to K⁺-free Krebs-Henseleit solution and high K⁺. Ouabain or exposure to K⁺-free Krebs-Henseleit solution inhibited acetylcholine or sodium nitroprusside-induced relaxation. KCl also inhibited the acetylcholine or sodium nitroprusside-induced relaxation. These results suggest that the Na⁺, K⁺-pump may play a role in endothelium-dependent acetylcholine-induced relaxation.