Pyrroline-5-carboxylate is the intermediate in the interconversions of proline, ornithine, and glutamate. Recent studies have suggested that pyrroline-5-carboxylate has regulatory properties in a wide range of tissues. Physiologic studies in humans have been limited by the unavailability of a sufficiently sensitive assay for this compound. Until now pyrroline-5-carboxylate has not been detectable in plasma of normal humans or even in plasma of patients with type II hyperprolinemia. We now report a method for measuring pyrroline-5-carboxylate made possible by a preparation of purified Escherichia coli pyrroline-5-carboxylate reductase. This method is more sensitive than currently available methods by two or three logs and is applicable for all biologic fluids. We have quantitated pyrroline-5-carboxylate in normal plasma, urine, and saliva. In addition, we have found pyrroline-5-carboxylate levels 10x to 20x normal in two patients with type II hyperprolinemia. The ability to measure physiologic concentrations of pyrroline-5-carboxylate now enables further characterization of its role as a regulatory molecule.