It has been demonstrated by other investigators that endothelium-dependent vasodilators are more effective on arterial tissue than on venous tissue. The purpose of this study was to determine if this was due to a difference in the sensitivity of arterial and venous smooth muscle to the endothelial dilator (EDRF) or to a difference in the ability of arterial and venous endothelia to release EDRF. To differentiate between these two possibilities, an in vitro “sandwich” preparation was used in which the mechanical response to endothelium-dependent dilators of a de-endothelialized vessel was determined when “sandwiched” with an endothelialized vessel. Using dog femoral artery and saphenous vein, it was determined that acetylcholine (ACh), the ionophore A23187, and thrombin were endothelium-dependent dilators of the femoral artery, but their dilatory ability was significantly less in the saphenous vein. However, if the de-endothelialized saphenous vein was “sandwiched” with an endothelialized femoral artery, both ACh and A23187 significantly relaxed the vein. No relaxation of the de-endothelialized femoral artery occurred when it was “sandwiched” with an intact saphenous vein. Sodium nitroprusside, thought to act by a mechanism similar to EDRF, relaxed equally the saphenous vein and femoral artery. These observations suggest that the difference in responsiveness between femoral arteries and saphenous veins to endothelium-dependent dilators is due more to differences in the ability of their endothelia to release EDRF than to an inability of their smooth muscle to respond to EDRF.