The chemical identification and functional characterization of endothelium-derived hyperpolarizing factors varies depending on vascular size, vascular bed and species. Three major candidates are the epoxyeicosatrienoic acids, cytochrome P450 metabolites of arachidonic acid, potassium ion and hydrogen peroxide. Additionally, electrical coupling through myoendothelial gap junctions serves to conduct electrical changes from the endothelium to the smooth muscle and may mediate or propagate hyperpolarization. Endothelium-derived hyperpolarizing factors are important mediators of vascular relaxation most specifically in resistance sized arteries where they regulate tissue blood flow. The release of the factors is modulated by a number of influences including agonist stimulation, shear stress, estrogen and disease. This article reviews the latest studies concerning the characterization of endothelium-derived hyperpolarizing factors, the mechanisms of factor release and alterations of the factors.