An aqueous extract of culture of Trichoderma viride Y244–2 on wheat bran showed antitumor activity against L5178Y mouse leukemic cells in vitro and L1210 mouse leukemia in vivo. The principle of the antitumor activity was proved to be an l-lysine-oxidizing enzyme. Oxygen consumption was accompanied by the formation of α-keto acid, ammonia and hydrogen peroxide in the enzyme reaction with l-lysine. The ratio of amount of oxygen consumed to those of α-keto acid and ammonia formed in the presence of catalase was approximately 1:2:2. This enzyme is an l-amino acid oxidase which is highly specific for l-lysine. Thus, we designated it as l-lysine oxidase. Identity of the antitumor substance with the enzyme was confirmed by DEAE-cellulose column chromatography: the elution pattern of growth-inhibitory activity against L5178Y cells coincided with that of l-lysine oxidase activity.