1. The vascular endothelium releases a hyperpolarizing factor (endothelium-derived hyperpolarizing factor, EDHF) tentatively identified as a cytochrome P450-derived arachidonic acid metabolite. However, there is still controversy concerning its transferability and identity. We designed a bioassay system for assessing EDHF release in which the membrane potential was recorded in cultured vascular smooth muscle cells located downstream from donor endothelial cells. 2. Under combined nitric oxide (NO) synthase and cyclo-oxygenase blockade with NG-nitro-L-arginine (100 mumol l-1) and diclofenac (10 mumol l-1), the superfusate from bradykinin (30 mumol l-1)-stimulated, cultured porcine coronary endothelial cells induced a distinct hyperpolarization followed by a depolarization. Direct application of bradykinin to the smooth muscle cells resulted solely in membrane depolarization. Similar results were obtained using bradykinin-stimulated porcine coronary arteries as donor. 3. Single-channel current measurements suggest that this EDHF-induced hyperpolarization was elicited by the activation of Ca(2+)-dependent K+ channels. 4. Increasing the transmural pressure within the donor segment significantly enhanced the duration, but not the amplitude of the hyperpolarization induced by the effluate from bradykinin-stimulated donor segments. 5. Inhibition of P450 oxygenase activity with clotrimazole (3 mumol l-1) or 17-octadecynoic acid (3 mumol l-1) abolished EDHF release from the coronary endothelium, while the P450-derived arachidonic acid metabolite, 5,6-epoxyeicosatrienoic acid, induced a hyperpolarization of detector smooth muscle cells almost identical to that induced by EDHF. Moreover, induction of P450 activity by beta-naphthoflavone (3 mumol l-1, 48 h), significantly increased the bradykinin-induced release of EDHF. 6. These findings suggest that the vascular endothelium releases a transferable hyperpolarizing factor, chemically distinct from NO and prostacyclin, in response to agonists and mechanical stimulation. This beta-naphthoflavone-inducible EDHF appears to be a cytochrome P450-derived metabolite of arachidonic acid, which elicits hyperpolarization by activation of Ca(2+)-dependent K+ channels.