Docosahexaenoic acid (DHA) shows more pronounced relaxation when blood vessel is contracted with prostanoid receptor agonists than other stimulants. The present study was carried out to obtain information on the mechanisms underlying prostanoid receptor-selective relaxant action of DHA, particularly focusing on the possible roles for K⁺ channels and its CYP epoxygenase (EOX) metabolites. In endothelium-denuded rat thoracic aorta, DHA (10⁻⁵ M ) almost completely relaxed U46619 (a thromboxane A₂ (TP) receptor agonist)-contracted muscle without substantially affecting noradrenaline (NA)-induced contraction. DHA-induced relaxation was not affected by a large conductance, calcium- and voltage-activated K⁺ (BK) channels inhibitor iberiotoxin (IbTX, 10⁻⁷ M ) but was almost abolished by high-KCl (8×10⁻² M ) or 10⁻² M tetraethylammonium (TEA) which non-selectively inhibits K⁺ channel activity. DHA also prominently relaxed U46619-contracted aorta even in the presence of CYP inhibitors (SKF525A or miconazole, each at 10⁻⁵ M ). However, in the presence of these CYP inhibitors, the relaxant action of DHA was not affected by 10⁻² M TEA. In supporting a significant role for CYP EOX metabolites in the blood vessel relaxation to DHA, 16,17-epoxy docosapentaenoic acid (16,17-EpDPE), but not 19,20-EpDPE, showed a potent relaxation in U46619-contracted aorta, and this action was significantly attenuated by 10⁻² M TEA. The present findings suggest that the relaxant action of DHA shown in the rat aorta contracted through the stimulation with TP receptor is generated by DHA itself and its CYP EOX metabolites. The relaxant effect of DHA metabolites seems to be partly triggered by the activation of K⁺ channels though the role for BK channel is insignificant.