Experiments were designed to investigate the importance of the endothelium in relaxation of isolated coronary arteries caused by beta-adrenoceptor agonists or adenosine. Rings of canine left circumflex coronary arteries were suspended for isometric tension recording in organ chambers filled with physiological salt solution. Norepinephrine, isoproterenol, adenosine, acetylcholine, and sodium nitroprusside caused relaxations of control rings contracted with prostaglandin F2 alpha or KCl. The relaxations to all substances tested were relatively smaller during contractions evoked by KCl than those caused by prostaglandin F2 alpha. Mechanical removal of the endothelium abolished the relaxations caused by acetylcholine, reduced those caused by the catecholamines and adenosine, and did not affect the relaxations to sodium nitroprusside during contractions to prostaglandin F2 alpha. Endothelium-removal did not affect relaxations to beta-adrenergic agonists or adenosine during contractions to KCl or after pretreatment of the arteries with indomethacin. Indomethacin did not affect responses to acetylcholine or sodium nitroprusside, but augmented those in response to beta-adrenoceptor stimulation and adenosine. These results suggest that the endothelial cells of the canine coronary artery produce a signal in response to beta-adrenergic agonists and adenosine, which facilitates the direct inhibitory action of these substances on vascular smooth muscle.