This study was aimed at defining the role of vascular endothelium in the transport and metabolism of adenosine. For this purpose, endothelium-intact and endothelium-denuded isolated rat aortas, perfused at constant flow (2 ml/min), were prelabled with3H-adenosine or3H-inosine for 10 minutes at concentrations of 0.012-100 μM. Sequestration of adenosine by endothelium was determined from radioactivity recovered during selective endothelial cell removal with deoxycholic add (0.75percent; for 15 seconds). In the physiological concentration range of adenosine (0.012- -1 üM), fractional sequestration by endothelium was 90–92percent; of the total adenosine incorporation by the aorta. Endothelial sequestration of inosine at 0.1 üM was 85percent;. At 100 üM adenosine or inosine, fractional sequestration by aortic endothelium was 33percent; and 39percent;, respectively. Analysis of the specific radioactivity of adenine nucleotides extracted from prelabeled aortas indicated that most of the adenosine was incorporated into endothelial adenine nudeotides. Incorporation of inosine into endothelial ATP was approximately 15percent; that of adenosine. Ininbition of aortic adenosine deaminase with erythro-9-(2-hydroxy-3-nonyI)adenine (EHNA) did not influence sequestration of 0.1 üM adenosine, but resulted in a 49percent; reduction of total endothelial incorporation at 100 üM adenosine. Transfer of radioactive purines from the endothelium to underlying smooth muscle after prelabeling was equivalent to only 1percent;/hr of total endothelial radioactivity. Our findings suggested that 1) macrovascular endothelium of the aorta constitutes a highly effective metabolic barrier for circulating adenosine and inosine; 2) transfer of labeled adenine nucleotides from endothelium to underlying smooth muscle is rather small and most likely proceeds via dephospborylated purine compounds; and 3) measurement of adenosine trapping in endothelial and smooth muscle compartments overestimates the transendothelial adenosine concentration gradient.