This study was designed to investigate the mediator(s) of endothelium-dependent arterial constrictor responses evoked by arachidonic acid in vitro. A segment of descending rabbit thoracic aorta was isolated and perfused (1-2 ml/min) with oxygenated Krebs' bicarbonate buffer. Changes in the vascular smooth muscle-contracting activity of the aortic effluent were detected by superfusion bioassay using either strips of rabbit aorta or rings of dog saphenous vein, both denuded of endothelium and exposed to indomethacin (10 microM). Arachidonic acid (5-50 micrograms) injected into the inflow of the perfused aorta caused a dose-related increase in the vascular smooth muscle-contracting activity of the aortic effluent, whereas arachidonic acid added directly into the aortic effluent did not. The arachidonic acid-induced elevation of vascular smooth muscle-contracting activity in the aortic effluent was not apparent when indomethacin (10 microM) was added to the aortic inflow to inhibit cyclooxygenase, when the endothelium of the perfused aorta was removed by rubbing, or when the thromboxane A2/prostaglandin H2 receptors of the vascular tissues used for bioassay were blocked with an antagonist (1 microM SQ29548), and was unaffected when an inhibitor of thromboxane synthase (10 microM CGS 13080) was added to the aortic inflow. This effect of arachidonic acid was accompanied by release of prostaglandin H2 (measured as prostaglandin F2 alpha after reduction with SnCl2) in amounts sufficient to elicit contraction of the vascular tissues used for bioassay and was attenuated when a reducing agent (2 mM FeCl2) that converts prostaglandin H2 to 12-heptadecatrienoic acid was added to the aortic effluent. Collectively, these observations suggest that arachidonic acid stimulates endothelium-dependent release from the perfused aorta of a prostanoid that contracts vascular smooth muscle via interaction with thromboxane A2/prostaglandin H2 receptors. The study also suggests that the prostanoid responsible for the vascular smooth muscle-contracting activity of the aortic effluent is a prostaglandin endoperoxide(s) rather than thromboxane A2.