We developed a radioisotopic assay for proline oxidase in which product deltal-pyrroline-5-carboxylate-14-C is reacted with o-aminobenzaldehyde and the radioactivity trapped as the dihydroquinazolinium compound is recovered by ion-exchange chromatography. The sensitivity of this method allows the measurement of proline oxidase activity in small specimens (10 to 20 mg.) of liver.