Abstract: Aim To investigate the role and mechanism of endothelium-derived hyperpolarizing factor (EDHF) in the vasorelaxation of rats. Methods Isometric tension was recorded in isolated rat aorta and small mesenteric arteries to study the effect of PGI₂, NO and EDHF on endothelium-dependent relaxation. Results The contribution of EDHF to endothelium-dependent relaxation is significantly larger in small mesenteric arteries than in the aorta. In the nitric oxide synthesis inhibited rats the contribution of EDHF transiently increased. The nature and vasodilating mechanism of EDHF may not be involved the cytochrome P450 pathway, while it is partially mediated by K_Ca channels. Conclusion In the small mesenteric artery of rats the endothelium-dependent relaxation was mainly caused by EDHF. When nitric oxide synthesis was inhibited, the contribution of EDHF increased in the early phase. The hyperpolarizing and vasodilating mechanism of EDHF is partially mediated by K_Ca channels.