An endothelium-derived hyperpolarizing factor (EDHF) mediates a part of the vasodilatory action of bradykinin. A bioassay method was developed to investigate the properties of EDHF on bovine coronary arterial smooth muscle cells. Cannulated bovine coronary arteries with an intact endothelium that were treated with indomethacin and N<sup>G</sup>-nitro- L-arginine methyl ester served as the EDHF donor. The effect of the donor vessel perfusate was examined on a 240 pS single-channel calcium (Ca<sup>2+</sup>)-activated potassium (K<sup>+</sup>) current (K<sub>Ca</sub>) and resting membrane potential in recipient coronary arterial smooth muscle cells. The open state probability (NPo) of the channel averaged 0.011 ± 0.003 during basal perfusate flow. After stimulation of the donor vessels with bradykinin (10<sup>–10</sup>–10<sup>–6</sup> M), the perfusate induced a 1.2- to 5-fold increase in the NPo (n = 7, p < 0.001). This increase in channel activity was attenuated by either removing the endothelium of the donor arterial segment or upon inhibition of cytochrome P<sub>450</sub> in the donor arterial segment with the combination of 17-octadecynoic acid and miconazole. The resting cell membrane averaged –60 ± 2 mV, and hyperpolarized to –69 ± 1.5 mV (n = 6, p < 0.05) in response to the perfusate following stimulation of the donor vessel with bradykinin. Addition of 14,15-epoxyeicosatrienoic acid mimicked the effects of the perfusate and increased the NPo of the K<sub>Ca</sub> channel from 0.01 ± 0.001 to 0.05 ± 0.001. These findings suggest that bradykinin stimulates the release of a transferable endothelial factor that activates K<sub>Ca</sub> channels and hyperpolarizes coronary arterial smooth muscle cell membranes. These findings support the hypothesis that coronary arteries release an EDHF which is a cytochrome P<sub>450</sub> metabolite of arachidonic acid.