The role of arachidonic acid in forming endothelium-derived relaxing factor remains controversial. This controversy may be explained if more than one factor exists. To test this hypothesis, the effects of various inhibitors of arachidonic acid metabolism were studied. The perfusate from canine femoral arteries with endothelium was bioassayed with coronary artery rings without endothelium. Treatment of the perfused segment (but not the bioassay ring) with inhibitors of phospholipase A2 (quinacrine) or cytochrome P450 (metyrapone) had no effect on the basal relaxing activity of the effluent; treatment with the inhibitor of lipoxygenase, nordihydroguaiaretic acid, significantly depressed it. With increasing concentrations of acetylcholine, a biphasic concentration-relaxation curve was obtained; the enzyme inhibitors depressed or prevented the first phase but did not affect the second phase. Infusion of arachidonic acid or soybean lipoxidase directly on the bioassay ring did not cause relaxation; together, they evoked concentration-dependent relaxations. These data suggest that acetylcholine can trigger the release of two chemically different relaxing mediators from the endothelium of the canine femoral artery. One factor may be a product of lipoxygenase (or epoxigenase). The second factor is not a metabolite of arachidonic acid and may be released under basal conditions. The existence of two (or more) chemically different endothelium-derived mediators may help to explain the controversial data regarding the nature of the factor(s).