Upon oxidation of D-propargylglycine by hog kidney D-amino acid oxidase, the enzyme is converted by covalent alkylation to catalytic species with different properties from those of native enzyme. At least 5 distinct modified enzyme species are present in the preparation, as determined by gel electrofocusing. Individual characterization of the components was not yet attempted. The combined kinetic and spectral properties of the preparation were studied. The modified enzymes have a marked preference for hydrophobic amino acids: the rates of oxidation decrease in the series D-phenylalanine, D-methionine, D-norleucine, D-norvaline, D-.alpha.-aminobutyrate, D-alanine. The observed Km of the amino acids are increased, especially those of the smaller substrates (D-alanine and D-.alpha.-aminobutyrate). A primary kinetic isotope effect is observed upon oxidation of amino acids by the modified enzymes, evidence that this catalysis exhibits a different rate-determining step from catalysis by native enzyme. The modified apoenzyme exhibits intense absorbance at 318-320 nm, not present in native enzyme. This chromophore can be partially (75%) removed by treatment of the modified enzyme with hydrazine. The activity of native enzyme is not substantially restored by this process, suggesting the existence of superficial alkylations in addition to the modification responsible for the observed changes in kinetic parameters.