Experiments were designed to investigate the importance of endothelial metabolism of arachidonic acid in the relaxation of isolated arteries caused by acetylcholine, ATP, arachidonic acid itself and thrombin. Rings of canine femoral arteries were mounted for isometric tension recording in organ chambers filled with Krebs-Ringer-bicarbonate solution. Acetylcholine, arachidonic acid, ATP, 15-hydroperoxy-5,8,11,13 eicosatetraenoic acid, thrombin and prostacyclin caused relaxations of control rings made to contract with norepinephrine. Removal of the endothelium abolished the relaxations caused by acetylcholine, ATP, thrombin and 15-hydroperoxy-5,8,11,13 eicosatetraenoic acid, reduced those caused by arachidonic acid, but did not affect the inhibitory effect of prostacyclin. The inhibitory response to arachidonic acid was abolished by indomethacin and 5,8,11,14-eicosatetraynoic acid (ETYA); that to acetylcholine was abolished by mepacrine and reduced by ETYA. The relaxations induced by thrombin and ATP were not affected by these inhibitors. Canine femoral arteries with endothelium, but not de-endothelialized preparations, transformed part of exogenously added [14C] arachidonic acid to prostaglandins (6-oxo-prostaglandin (F1 alpha) and a hydroxy derivative. The formation of prostanoids was inhibited by indomethacin, ETYA and 15-hydroperoxy-5, 8,11,13 eicosatetraenoic acid and that of hydroxy derivative by ETYA. These results suggest that the endothelial cells of canine femoral arteries initiate relaxation of the vascular smooth muscle cells of the media by: 1) producing prostacyclin, when exposed to arachidonic acid; 2) producing a lipoxygenase product, when exposed to acetylcholine; and 3) producing a signal of unknown nature, when exposed to thrombin or ATP.