The present study demonstrates that rabbit aortic intimal smooth muscle cells contain the majority of their endogenous arachidonic acid mass in plasmenylethanolamine molecular species. To demonstrate the potential significance of these plasmenylethanolamines as substrates for the smooth muscle cell phospholipases that are activated during agonist stimulation, aortic rings were prelabeled with [3H]arachidonic acid and stimulated with angiotensin II. Although the specific activities of the choline and inositol glycerophospholipid pools were similar after the labeling interval, ethanolamine glycerophospholipids had a specific activity of only 20% of the specific activity of choline and inositol glycerophospholipids. Despite the marked disparity in the specific activities of these three phospholipid classes, angiotensin II stimulation resulted in similar fractional losses (35-41%) of [3lljarachidonic acid from vascular smooth muscle choline, ethanolamine, and inositol glycerophospholipid classes. Reverse-phase HPLC demonstrated that >60% of the [3H]arachidonic acid released from ethanolamine glycerophospholipids after angiotensin II stimulation originated from plasmenylethanolamine molecular species. Taken together, the results demonstrate that the major phospholipid storage depot for arachidonic acid in vascular smooth muscle cells are plasmenylethanolamine molecular species which are important substrates for the phospholipase(s) that are activated during agonist stimulation.Icosanoids are important regulators of vascular smooth muscle cell contractile state, hypertrophy, and proliferation (1-6). Accordingly, significant attention has focused on the biochemical events responsible for the liberation of arachidonic acid from endogenous smooth muscle cell lipids during signal transduction. Arachidonic acid release from cellular phospholipids may occur directly in a single catalytic step by the action of phospholipase A2 or by a multistep sequential pathway initiated by phospholipase C and culminated by the sequential actions of diglyceride and monoglyceride lipases (7)(8)(9). Although recent studies demonstrated that radiolabeled arachidonic acid was released from choline, ethanolamine, and inositol glycerophospholipid in cultured smooth muscle cell pools after agonist stimulation, quantification of the amount of released radiolabeled arachidonic acid from each pool suggested that different phospholipid classes were primarily responsible for radiolabeled arachidonic acid release (10-13). Since the biochemical and physiologic properties of cultured smooth muscle cells differ substantially from properties of vascular smooth muscle cells in intact tissue, the present study was performed to assess the distribution of arachidonic acid mass in phospholipids and the fractional turnover of individual phospholipid molecular species in intact vascular smooth muscle. The results demonstrate that the majority of arachidonic acid mass in vascular smooth muscle is present in plasmenylethanolamine molecular species and that ...