1. Melatonin (10(-4)-10(-3) M) inhibited contractile response to 5-hydroxytryptamine (5-HT) and KCl in rat isolated aorta. 2. In the presence of verapamil but not nifedipine, melatonin failed to inhibit residual response to KCl. 3. In the aorta precontracted with 5-HT, PGF2 alpha, or KCl, melatonin (10(-6)-10(3) M) caused relaxation. Removal of endothelium only inhibited the melatonin relaxation on the 5-HT response. Methylene blue also inhibited the melatonin relaxation. 4. Nifedipine and verapamil partly inhibited the melatonin relaxation on the 5-HT response. In the absence of endothelium, verapamil but not nifedipine further inhibited the melatonin relaxation. 5. M & B 22,948 inhibited the melatonin relaxation. Melatonin potentiated the nitroglycerin relaxation. In the absence of endothelium, nitroglycerin and melatonin potentiated the relaxation by melatonin and nitroglycerin, respectively. 6. These results suggest that the mode of inhibitory action of melatonin is somewhat similar to that of verapamil. In addition, the vasorelaxing effect of melatonin on the 5-HT response is endothelium dependent and also may be related to the inhibition of cGMP metabolism.