The mechanism of agonist-activated arachidonate release was studied in segments of rat tail artery. Tail artery segments were prelabeled with [³H]arachidonate and then stimulated with norepinephrine (NE), and the radioactivity of the extracellular medium was determined. NE stimulated arachidonate release from the tissue without increasing arachidonic acid levels within cellular cytosol or crude membranes. About 90% of the extracellular radioactivity was shown to be unmetabolized arachidonate by TLC. Arachidonic acid release was not inhibited by the removal of the endothelium from the artery. NE exerted a half-maximal effect at a concentration of 0.2 µM. NE-stimulated arachidonate release was not inhibited by blockers of phospholipase C (U-73122), diacylglycerol lipase (RHC-80267), secretory phospholipase A₂ (manoalide), calcium-insensitive phospholipase A₂ (HELSS), or -adrenergic receptors (propranolol). NE-stimulated arachidonic acid release was inhibited by blockers of cytosolic phospholipase A₂ (cPLA₂) (AACOCF₃), ₁-adrenergic receptors (prazosin), and specific G proteins (pertussis toxin). This indicated that NE stimulated arachidonate release from vascular smooth muscle via activation of -adrenergic receptors, either G_i or G_o, and cPLA₂. NE-activated arachidonic acid release from vascular smooth muscle may play a role in force generation by the tissue. Perhaps arachidonic acid extends the effect of NE on one specific smooth muscle cell to its nearby neighbor cells.