Arachidonic acid elicited relaxation responses in normal rabbit aorta precontracted with norepinephrine. The relaxation response was enhanced by the cyclooxygenase inhibitor indomethacin and inhibited by lipoxygenase inhibitors, including nordihydroguaiaretic acid and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. The cytochrome P-450 epoxygenase inhibitor metyrapone had no effect on arachidonic acid-induced relaxations. The present study hypothesized that a lipoxygenase metabolite of arachidonic acid mediated the response. Incubation of rabbit aorta with [14C]arachidonic acid resulted in the synthesis of a previously unidentified 14C-labeled metabolite and was called the unknown factor. Production of the unknown factor was not inhibited by indomethacin and decreased by lipoxygenase inhibitors. Production of the unknown factor and arachidonic acid-induced relaxations were dependent on an intact endothelium, indicating that the cellular source of the unknown relaxant factor was the endothelial cell. This was confirmed by demonstrating the ability of cultured rabbit aortic endothelial cells to produce the unknown factor from [14C]arachidonic acid. Feeding rabbits a 2% cholesterol diet for 2 wk induced hypercholesterolemia without causing atherosclerosis. In the cholesterol-fed rabbits, indomethacin enhanced arachidonic acid-induced relaxations in norepinephrine-precontracted aortas (maximal relaxation 49.0 +/- 2.5 vs. 35.5 +/- 1.7%, cholesterol-fed vs. normal) and increased production of the unknown factor compared with normal rabbits. The partially purified unknown factor elicited an approximately 26% inhibition of the vasoconstrictor response to norepinephrine in intact rabbit aorta. Further purification of the unknown factor by reverse-phase high-pressure liquid chromatography system resulted in isolation of a radioactive product that relaxed precontracted rabbit aorta. Therefore these data suggest that in normal and hypercholesterolemic rabbit aorta the endothelium produces an unknown metabolite of arachidonic acid that causes vasorelaxation and may regulate vascular tone.