A novel enzyme responsible for the oxidation of spermidine and spermine has been found in rat liver. Spermidine is shown to be degraded to putrescine and 3-aminopropionaldehyde, and spermineto be cleaved to spermidine and 3-aminopropionaldehyde. A single enzyme catalyzing both reactions and designated as polyamine oxidase has been pu-rified 4000-fold to electrophoretic homogeneity. Polyamine oxidase appears to be a flavoprotein, containing flavin adenine dinucleotide (FAD) as a prosthetic group. Hydrogen peroxide is evolved in the reaction and no other electron acceptors except molecular oxygenhave been found. The molecular weight of the enzyme was approximately 60 000 and the sedimentation coefficient 4.5 S. The enzyme appears to be a single polypep-