Determination of the amino acid sequence in the active site region of chicken liver L-2-hydroxyacid oxidase A after limited proteolysis of the polypeptide chain clearly establishes that the enzyme responsible for the oxidative decarboxylation activity is a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family. A fingerprint of the NAD(P)H binding domain, shows that the chicken liver oxidase present a high level of similarity with the malic enzyme family.