Rat aortic endothelium is differentiated regionally for three signal pathways capable of regulating the cGMP content of the underlying smooth muscle. Formation of nitric oxide (NO) from L-arginine and of glutamate from L-leucine increase cGMP; however, formation of prostaglandin H₂ (PGH₂) decreases cGMP. All three have peak activity in the windkessel area just distal to the aortic arch and decrease peripherally. We report evidence that the biochemical route of the leucine-to-glutamate (LeuGlu) pathway is via metabolism of leucine to acetyl CoA, that the controlling reaction of the pathway is mediated by the branched chain -ketoacid dehydrogenase complex (BCDC), and that glutamate formation via the LeuGlu pathway is a major source of aortic segment free glutamate in vitro. Interruption of the pathway by treatment of precontracted rat aortic rings in vitro with each of three classes of inhibitors (leucine analogs, competitors for the BCDC reaction, or inhibitors of L-glutamate transport) enhances contractile responses. The enhancement requires an intact endothelium and is not owing to reductions in NO formation. The results support the hypothesis that the LeuGlu pathway functions in the regulation of aortic contractility and compliance.