The aim of the present study was to assess whether or not thrombin affects vascular smooth muscle activity by a direct action on the blood vessel wall. In isolated saphenous veins and femoral and pulmonary arteries thrombin did not affect basal tension, but in pulmonary and portal mesenteric veins it caused contraction. In saphenous and femoral veins and in femoral and pulmonary arteries the enzyme depressed contractions evoked by α-adrenergic stimulation. In the saphenous vein thrombin also depressed contractions induced with acetylcholine or potassium ions. In this blood vessel the relaxation caused by thrombin was abolished by antithrombin III and the tripeptide D-phenylalanine-proline-arginine-CH<sub>2</sub>Cl, a specific inhibitor of the enzymatic center of thrombin. In pulmonary and portal mesenteric veins, thrombin potentiated the contractile response to norepinephrine. These contractile effects of thrombin were also abolished by D-phenylalanine-proline-arginine-CH<sub>2</sub>Cl. Thus, thrombin causes relaxation or contraction of blood vessels through a direct action on the blood vessel wall. These effects are due to its proteolytic activity.