The leucine-to-glutamate(Leu-Glu)pathway, which metabolizes the carbon atoms of L-leucine to form L-glutamate, was studied by incubating rat tissue segments with L-[U-¹⁴C]leucine and estimating the [¹⁴C]glutamate formed. Metabolism of the leucine carbon chain occurs in most rat tissues, but maximal activity of the Leu->Glu for glutamate formation is limited to the thoracic aorta and pancreas. In rat aorta the Leu->Glu functions to relax the underlying smooth muscle; its functions in pancreas are unknown. This report characterizes the Leu->Glu of rat pancreas and develops methods to examine its functions. Pancreatic segments effect net formation of glutamate on incubation with L-leucine, L-glutamine or a mix of 18 other plasma amino acids at their concentrations in rat plasma. Glutamate formed from leucine remains mainly in the tissue, whereas that from glutamine enters the medium. The pancreatic Leu->Glu pathway uses the leucine carbons for net glutamate formation, the alpha-amino group is not used; the stoichiometry is 1 mole of leucine yields 2 moles of glutamate (2 leucine carbons per glutamate) plus 2 moles of CO₂. Comparison of the Leu->Glu pathway in preparations of whole pancreatic segments, isolated acini, and islets of Langerhans localize it in the acini; relatively high activity is found in cultures of the AR42J cell line and very little in the INS-1 832/13 cell line. Pancreatic tissue glutamate concentration is homeostatically regulated in the range of 1-3 umol/g wet weight. L-valine and leucine ethyl, -benzyl, and -ter-butyl esters inhibit the Leu->Glu without decreasing total tissue glutamate.