In vascular tissues, many types of stimulants which release endothelium-derived relaxing factor (EDRF) hyperpolarized the smooth muscle membrane in an endothelium-dependent manner. The hyperpolarizations were resistant to inhibitors of the actions of EDRF, thereby suggesting that they were elicited by an unidentified substance called endothelium-derived hyperpolarizing factor (EDHF). The EDHF induced hyperpolarization was produced by an increase in K(+)-permeability of the membrane, and was one of the factors to induce endothelium-dependent vasodilations. Release of EDHF required an increase in endothelial Ca2+, due either to release of Ca2+ from intracellular storage or to its influx from the external media.