Airway smooth muscle (ASM) metabolizes arachidonic acid through various enzymatic pathways, including cytochrome P-450 (CYP-450) -hydroxylase, which leads to the production of 20-hydroxy-eicosatetraenoic acid (20-HETE). The goal of this study was to delineate the mode of action of 20-HETE in human ASM cells. Isometric tension measurements demonstrated that 20-HETE induced a concentration-dependent relaxant effect in ASM on bronchi precontracted with either MCh or arachidonic acid (AA). Relaxing effects of 20-HETE on resting tone were prevented by 10 nM Iberiotoxin (IbTx), a BK_Ca channel inhibitor. Microelectrode measurements showed that exogenous additions of 20-HETE (0.1-10 µM) hyperpolarized the membrane potential of human ASM cells. This concentration-dependent electrophysiological effect induced by the eicosanoid was prevented by 10 nM IbTx. Complementary experiments, using the planar lipid bilayer reconstitution technique, demonstrated that 20-HETE activated reconstituted BKCa channels at low free Ca²⁺ concentrations. Altogether these results indicate that 20-HETE-dependent activation of BK_Ca channels is responsible for the hyperpolarization and controlled relaxation of ASM in human distal bronchi.