Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in endothelial cells is proposed to be required for generation of vascular actions of endothelium-derived hyperpolarizing factor (EDHF). This study was designed to determine the endothelial Ca²⁺ source that is important in development of EDHF-mediated vascular actions. In porcine coronary artery precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid (CPA) caused endothelium-dependent relaxations in the presence of N^G-nitro-L-arginine (L-NNA). The L-NNA-resistant relaxant responses were inhibited by high K⁺, indicating an involvement of EDHF. In the presence of Ni²⁺, which inhibits Ca²⁺ influx through nonselective cation channels, the BK-induced EDHF relaxant response was greatly diminished and the CPA-induced response was abolished. BK and CPA elicited membrane hyperpolarization of smooth muscle cells of porcine coronary artery. Ni²⁺ suppressed the hyperpolarizing responses in a manner analogous to removal of extracellular Ca²⁺. EDHF-mediated relaxations and hyperpolarizations evoked by BK and CPA in porcine coronary artery showed a temporal correlation with the increases in [Ca²⁺]_i in porcine aortic endothelial cells. The extracellular Ca²⁺-dependent rises in [Ca²⁺]_i in endothelial cells stimulated with BK and CPA were completely blocked by Ni²⁺. These results suggest that Ca²⁺ influx into endothelial cells through nonselective cation channels plays a crucial role in the regulation of EDHF.