An assay procedure for polyamine oxidase in tissue homogenates was devised. The method is based on the degradation of N1,n12-diacetylspermine to N1-acetylspermidine and the determination using TLC of the latter. Polyamine oxidase activity is high in most tissues. Its activity is comparable to that of spermidine and spermine synthase. The independence of this enzyme from cellular proliferation rates and its relatively long biological half-life are indicative of a passive role of polyamine oxidase in the regulation of cellular polyamine levels.