Although endothelium-derived hyperpolarizing factor (EDHF) activity has been demonstrated in arteries from various species, EDHF has not been chemically identified, nor its mechanism of action characterized. To elucidate this mechanism, we tested the effect of EDHF on large-conductance Ca2+-activated K+ (KCa) channels in porcine coronary artery smooth muscle cells. By using a patch-clamp technique, single-channel currents were recorded in cultured smooth muscle cells; the organ bath also contained a strip of porcine coronary with endothelium, which served as the source of endothelium-derived relaxing factor(s) including EDHF. Exposure of endothelium to 10−6M bradykinin activated KCa channels in cultured smooth muscle cells in cell-attached patches. When the experiment was performed in the presence of 10 μM indomethacin and 30 μM NG-nitro-L-arginine (L-NNA), which block the generation of prostaglandin I2 (PGI2) and NO, respectively, KCa channel activity was stimulated by bradykinin, indicating the direct involvement of EDHF in KCa channel stimulation. Neither 10 μM methylene blue nor 25 μM Rp-cAMPS inhibited bradykinin-induced KCa channel activity. In inside-out patches, the addition of bradykinin to the solution was without effect on KCa channel activation. However, in the presence of 0.5 mM guanosine triphosphate (GTP) and 1.0 mM adenosine triphosphate (ATP) in the bath solution, KCa channels was activated by bradykinin. In outside-out patches, the addition of bradykinin also increased KCa channel activity, when GTP and ATP were added to the pipette solution. The addition of GDP-β-S (100 μM) in the cytosolic solution completely blocked the activation KCa channels induced by bradykinin in inside-out and outside-out patches. Pretreatment with 30 μM quinacrine, a phospholipase A2 inhibitor, or 3 μM 17-octadecynoic acid (17-ODYA), a cytochrome P450 inhibitor, in addition to indomethacin and L-NNA, abolished bradykinin-stimulated KCa channel activity in cell-attached patches. Both 14,15-epoxyeicosatrienoic acid (EET) and 11,12-EET increased the open probabilities of KCa channels in cell-attached patches. These results suggest that EDHF, released from endothelial cells in response to bradykinin, hyperpolarizes smooth muscle cells by opening KCa channels. Furthermore, our data suggest that EDHF is an endothelium-derived cytochrome P450 metabolite of arachidonic acid. The effect of EDHF on KCa channels is not associated with an increase of cAMP and cGMP. The activation of KCa channels appears to be due to the activation of GTP-binding protein.