1. Experiments were designed to determine the role of the endothelium in relaxations of isolated blood vessels caused by ACh, adenine nucleotides and K+ ions. 2. Paired rings of canine femoral arteries were mounted for isometric tension recording in organ chambers filled with aerated Krebs-Ringer solution (37 degrees C). One ring served as control; in the other ring the intimal layer was removed mechanically. 3. Removal of the endothelium only slightly depressed the maximal contractile response to noradrenaline, and did not affect the apparent sensitivity to the catecholamine. It depressed the contractile response to 25-60 mM-K+ more than that to noradrenaline. 4. In the absence of endothelium, the femoral arteries did not relax on exposure to ACh. 5. Removal of the endothelium did not affect relaxations caused by adenosine and AMP, but markedly reduced those caused by ADP and ATP. 6. The relaxations induced by 5.9 mM-K+ were comparable in control rings and arteries denuded of their endothelium. 7. These experiments demonstrate that in the canine femoral artery, relaxations induced by ACh, ADP and ATP require the presence of functional endothelial cells, which, when exposed to these substances, initiate an inhibitory signal for the smooth muscle cells of the media. By contrast, relaxations of isolated arteries caused by adenosine, AMP and K+ ions must be due mainly to a direct effect on the vascular smooth muscle cells.