Human cytochrome P-450 epoxygenase enzymes metabolize eicosapentaenoic acid (EPA), an 3-poly unsaturated fatty acid (PUFA), and leads to the production of 17(18)-epoxyeicosatetraenoic acid or 17(18)-EpETE. The aim of the present study was to delineate the mode of action of 17(18)-EpETE on human pulmonary artery (HPA) and distal bronchi. Isometric tension measurements demonstrated that 17(18)-EpETE induced concentration-dependent relaxing effects in pulmonary artery and airway smooth muscles. Iberiotoxin (IbTx) and Glyburide (Glyb), known BK_Ca and K_ATP channel inhibitors respectively, reversed the relaxation induced by 17(18)-EpETE on both tissues types. Microelectrode measurements showed that exogenous addition of 17(18)-EpETE hyperpolarized the membrane potential of HPA and bronchial smooth muscle cells. These induced electrophysiological effects were reversed by the addition of 10 nM IbTx and 10 µM Glyb. Complementary experiments performed on human bronchi, using the planar lipid bilayer reconstitution technique, demonstrated that 17(18)-EpETE activated reconstituted BK_Ca channels at low free Ca²⁺ concentration. Moreover in bronchi, the relaxing responses induced by 17(18)-EpETE were also related to reduced Ca²⁺ sensitivity of the myofilaments, since free Ca²⁺ concentration-response curves, performed on -escin permeabilized cultured explants, were shifted toward higher [Ca²⁺]. Together these results provide new insight into the mode of action of 17(18)-EpETE in lung tissues and highlight this eicosanoid as a potent modulator of tone on both HPA and distal bronchi in vitro, which may be of clinical relevance in the pathophysiology of pulmonary hypertension and airway diseases.