The aim of this study was to delineate the mode of action of 20-hydroxy-eicosatetraenoic acid (20-HETE) in airway smooth muscle (ASM) cells. ASM metabolizes arachidonic acid by various enzymatic pathways, including the CYP-450 -hydroxylase, which leads to the production of 20-HETE, a bronchoconstrictive eicosanoid. The present study demonstrated that 20-HETE induced concentration-dependent tonic responses in airway smooth muscle, whereas transient responses were recorded in Ca²⁺ free solution, suggesting an intracellular Ca²⁺ release process (8). 20-HETE inotropic responses were abolished by either 36 µM 2-APB or 1µM thapsigargin, but were insensitive to 10 µM ryanodine indicating that InsP3 receptors likely control the release of intracellular Ca²⁺. Sustained tension, which required Ca²⁺ entry, was partially blocked by 1 µM nifedipine (an L-type) and 100 µM Gd³⁺ (a non-selective cationic channel blocker). Moreover, in the absence of selective 20-HETE-receptor antagonists, 20-HETE tonic responses were inhibited in a concentration-dependent manner (0.1-10 µM) by capsazepine, a well-characterized vanilloid receptor antagonist. Capsazepine was also observed to reverse cumulative responses to 20-HETE and capsaicin, a TRPV1 agonist. In addition, capsazepine pretreatment largely modified the sustained inotropic responses to 20-HETE, suggesting that 20-HETE either cross-reacted with TRPV1 receptors with a low affinity (µM) or that its specific receptor was inhibited by the vanilloid antagonist. Data obtained using RHC-80267, ONO-RS-082 and Eicosatetraynoic Acid respective inhibitors of DAG-lipase, Phospholipase A2 and CYP-450 -hydroxylase, reveal that intracellular arachidonic acid production and its 20-HETE metabolite may be responsible for the activation of non-selective cationic channels and tonic responses.