In cascade perfusion and superfusion experiments on rabbit tissues, when acetylcholine (ACh) was introduced into the circuit so as to perfuse the aorta under perfusion with noradrenaline (NA), the effluent relaxed the transverse aortic strip which had been denuded of endothelium. The effluent from the perfused aorta which was capable of relaxing the transverse aortic strip also significantly inhibited platelet aggregation induced by arachidonic acid (AA) in a volume-related manner. The inhibitory activity was decreased by the prolongation of transit time before addition of the effluent to platelet-rich plasma. Neither the inhibition of AA-induced aggregation nor the relaxation of the transverse strip by the effluent could be observed after the removal of endothelium from the aorta, or after pretreatment of aorta with mepacrine or nordihydroguaiaretic acid (NDGA). The AA-induced platelet aggregation was unaffected by pretreatment of platelets with mepacrine or NDGA at the concentration tested. Pretreatment of aorta with indomethacin failed to modify the relaxation of the transverse strip induced by the effluent. These results strongly suggest that endothelium-derived vascular relaxant factor (EDRF) possesses inhibitory activity on AA-induced aggregation in addition to its vasodilator activity.