L-Lysine alpha-oxidase from Trichoderma viride Y244-2 has been purified to homogeneity. The enzyme shows absorption maxima at 277, 388, and 466 nm and a shoulder around 490 nm and contains 2 mol of FAD/mol of enzyme. The enzyme has a molecular weight of approximately 116,000 and consists of two subunits identical in molecular weight (about 56,000). In addition to L-lysine, L-ornithine, L-phenylalanine, L-tyrosine, L-arginine, and L-histidine are oxidized by the enzyme to a lesser extent. Several lysine analogs such as delta-hydroxylysine are oxidized efficiently. Balance studies showed that 1 mol of L-lysine is converted to an equimolar amount of alpha-keto-epsilon-aminocaproate, ammonia, and hydrogen peroxide with the consumption of 1 mol of oxygen. alpha-Keto-epsilon-aminocaproate spontaneously is dehydrated intramolecularly into delta 1-piperideine-2-carboxylate in the presence of catalase, and is oxidatively decarboxylated into delta-aminovalerate in the absence of catalase. The Michaelis constants are as follows: 0.04 mM for L-lysine, 0.44 mM for L-ornithine, 14 mM for L-phenylalanine, and 1.6 mM for oxygen with L-lysine.