Amiloride inhibits vascular smooth muscle contractions from canine aorta and saphenous vein. The mechanisms were studied using radioligand binding and functional techniques. Amiloride inhibited [3H]prazosin and [3H]rauwolscine binding to alpha-1 and alpha-2 adrenoceptors in a concentration-dependent manner. Amiloride increased Kd values for [3H]rauwolscine without affecting the maximum binding of [3H]prazosin. These results suggest that the drug interacts with the alpha-1 adrenoceptor binding sites in a competitive manner and with the alpha-2 adrenoceptor binding sites in a noncompetitive manner. Amiloride reduced maximal contractile responses to agonists selective for both alpha adrenoceptors and to elevated K+, the EC50 values were increased by about 10-fold in the presence of amiloride. In Ca+(+)-free Krebs' solution, contractions induced in saphenous vein after addition of Ca++ in saphenous vein in the presence of adrenoceptor agonists were inhibited by amiloride. Our results suggest that amiloride reduced alpha-1 and alpha-2 adrenoceptor-mediated responses and inhibited Ca++ influx.