The endothelial cells inhibit the tone of the underlying vascular smooth muscle by releasing endothelium-derived relaxing factors (EDRF). The existence of at least two such factors, nitric oxide and endothelium-derived hyperpolarizing factor (EDHF), has been demonstrated. EDHF is an as yet unidentified substance that hyperpolarizes vascular smooth muscle cells and causes their relaxation. The contribution of endothelium-dependent hyperpolarization varies along the vascular tree. Particularly in smaller blood vessels, EDHF acts on vascular smooth muscle in cooperation with nitric oxide. Basal release of EDHF is not likely to occur, at least in vitro. The production and/or release of EDHF is regulated by the cytosolic concentration of Ca2+ ions, derived both from the extracellular space and intracellular stores. Calmodulin may be involved in its production and/or release. EDHF hyperpolarizes the vascular smooth muscle by opening K+ channels. The hyperpolarization closes voltage-dependent Ca2+ channels and, as a consequence, EDHF relaxes blood vessels. In the absence of chemical identification of EDHF, it is difficult to assess its contribution to endothelium-dependent relaxations in vivo.