Endothelium-derived hyperpolarizing factor (EDHF) mediates vasodilatation in certain blood vessels, together with prostacyclin and NO. However, its chemical nature is not known. A perfusion-superfusion cascade was developed to confirm the diffusible nature of EDHF. Canine carotid arteries with endothelium were used as donors of vasoactive substances, whereas rings of coronary artery without endothelium were used as detectors. Inhibitors of NO synthesis and cyclooxygenase were present throughout, to avoid interference from NO and prostanoids. Measurements of membrane potential and isometric tension, in coronary arteries without endothelium (used as detectors), demonstrated the release of EDHF from the carotid arteries, following treatment with 8-methoxypsoralen, bradykinin and thimerosal. The K+-channel blocker tetraethylammonium inhibited the action od EDHF in the detectors. Thus, these results demonstrate that endothelial cells release a diffusible activator of K+-channels in vascular smooth muscle.