These studies test the hypothesis that L-glutamine at its physiological plasma concentration, ~0.5 mM, can increase tissue content and net synthesis of glutamate in rat aortic segments in vitro, thereby mediating relaxation of the underlying smooth muscle in the elastic reservoir region of the thoracic aorta. Aortic segments were incubated in an isotonic medium with and without 21 amino acids at their normal plasma concentrations. Of these amino acids only L-glutamine or L-leucine at their plasma concentrations increased glutamate synthesis and content. Tissue glutamate content resulting from increasing concentrations of each precursor reached an upper level of ~1.3-1.6 µmol/g wet weight. Regulation of the tissue glutamate content involves an interaction of the synthetic pathways in which L-glutamine inhibits the endothelial leucine-to-glutamate pathway. L-glutamine increases nitric oxide formation and NO inhibits the controlling enzyme of the endothelial leucine-to-glutamate pathway, the branched-chain -ketoacid dehydrogenase complex. Treatment of precontracted aortic rings with 0.5 mM L-glutamine elicits smooth muscle relaxation, a response which requires eNOS activity and an intact endothelium. The results demonstrate that in vitro L-glutamine at its normal concentration in plasma can regulate rat aortic glutamate content and modulate NO formation and contractility responses of the thoracic aortic wall.