1. Substrate specificity of D-amino acid oxidase was examined in the kidney homogenates of the mouse and the six species of fishes. 2. The enzyme of the mouse did not show a significant intraspecies variation in the substrate specificity. The sex and age of the mouse did not affect the substrate specificity of the enzyme. 3. The degree of similarity in the substrate specificity of the enzymes was quantified as the variation index. The values of the variation index among the enzymes of the fishes paralleled their taxonomic relatedness.