Because alterations in the aortic metabolism of arachidonic acid and in vascular responsiveness occur in hypercholesterolemic rabbits, we hypothesized that an arachidonic acid metabolite may contribute to the regulation of vascular tone. Aortic contractions to norepinephrine were investigated in rabbits fed either standard chow or chow containing 2% cholesterol. In normal rabbits, norepinephrine (10(-6) M) elicited a 126 +/- 2% contraction compared with a 95 +/- 2% contraction in cholesterol-fed rabbits. The factor mediating the depressed response was endothelium-dependent because removal of the endothelium blocked the decrease in norepinephrine-induced contractions observed in the cholesterol-fed rabbits. The endothelium-derived factor was not nitric oxide, because blockade of nitric oxide synthase with nitro-L-arginine did not abolish the decreased response in the cholesterol-fed rabbits. Pretreatment of aortas with a cyclooxygenase inhibitor, indomethacin (10(-5) M) caused a slight decrease in the norepinephrine-induced contractions, suggesting that the factor could be a vasoconstrictor cyclooxygenase metabolite or a vasodilatory lipoxygenase or cytochrome P450 epoxygenase metabolite. Pretreatment with the thromboxane A2/prostaglandin H2-receptor antagonist, SQ 29458, had no effect on norepinephrine-induced contractions. Whereas the lipoxygenase inhibitor, nordihydroguaiaretic acid (5 x 10(-5) M), caused a slight increase in the contractions to norepinephrine in cholesterol-fed rabbits compared with normal rabbits, the cytochrome P450 epoxygenase inhibitor, metyrapone (10(-4) M), produced a greater enhancement of norepinephrine-induced contractions in cholesterol-fed rabbits but had no effect on responses in the normal rabbits. Characterization of [3H]arachidonic acid metabolism in cholesterol-fed aortic tissue indicated that norepinephrine stimulated the synthesis of both lipoxygenase and epoxygenase metabolites in an endothelium-dependent manner. This study demonstrated that (a) an endothelium-derived metabolite of arachidonic acid regulates vascular tone, (b) this metabolite appears to be a lipoxygenase or cytochrome P450 product or both, and (c) the activity or synthesis of the factor is enhanced by hypercholesterolemia.