D-Amino acid oxidase catalyzes the oxidation of D-amino acids to imino acids with subsequent transfer of the electrons to molecular oxygen. Proposed mechanisms for the mode of cleavage of the substrate CH bond include stepwise formation of a carbanion, followed by attack of the carbanion on the enzyme-bound FAD, direct hydride transfer of the substrate alpha-hydrogen to the FAD, and transfer of a hydride from the substrate amino group to the FAD. Conditions have previously been established under which large, limiting, primary deuterium kinetic isotope effects can be measured with D-alanine, D-serine, and glycine as substrates for D-amino acid oxidase [Denu, J. M., & Fitzpatrick, P. F. (1992) Biochemistry 31, 8207-8215]. To determine whether these values are the intrinsic isotope effects, primary tritium kinetic isotope effects have been determined with these three substrates. The values are 12.6, 8.6, and 6.4, respectively. These values are consistent with expression of the intrinsic isotope effects under these conditions, allowing for determination of the values of the intrinsic deuterium effects as 5.7, 4.5, and 3.6 for D-alanine, D-serine, and glycine, respectively. Under these conditions, the alpha-secondary tritium kinetic isotope effect with glycine, the beta-secondary deuterium kinetic isotope effect with D-alanine, and the solvent kinetic isotope effect with D-serine are all indistinguishable from unity. These results are not consistent with concerted mechanisms for CH bond cleavage with this enzyme, but are fully consistent with the involvement of a carbanion intermediate.