Rabbit airway tissue is a particularly rich source of cytochrome P-4504A protein, but very little information regarding the effect(s) of 20-hydroxyeicosatetraenoic acid (20-HETE) on bronchial tone is available. Our studies examined the response of rabbit bronchial rings to 20-HETE and the metabolism of arachidonic acid and 20-HETE from airway microsomes. 20-HETE (10(-8) to 10(-6) M) produced a concentration-dependent relaxation of bronchial rings precontracted with KCl or histamine but not with carbachol. Relaxation to 20-HETE was blocked by indomethacin or epithelium removal, consistent with the conversion of 20-HETE to a bronchial relaxant by epithelial cyclooxygenase. A cyclooxygenase product of 20-HETE also elicited relaxation of bronchial rings. [14C]arachidonic acid was converted by airway microsomes to products that comigrated with authentic 20-HETE (confirmed by gas chromatography-mass spectrometry as 19- and 20-HETE) and to unidentified polar metabolites. [3H]20-HETE was metabolized to indomethacin-inhibitable products. These data suggest that 20-HETE is an endogenous product of rabbit airway tissue and may modulate airway resistance in a cyclooxygenase-dependent manner.