D-Amino acid oxidase was shown to dissociate into subunits in 2 M urea retaining the catalytic activity. This makes possible the direct observation of ESR spectra of the intermediate radical state of the enzyme when interacting with the substrate. We have shown that these radicals are really observable. Using the reversibility of the reaction and an equilibrium shift the amount of radicals can be increased up to 10% of all flavin groups present. The dependence of the radicals concentration on the amount of substrate and product can be predicted. The theory is confirmed by experimental data.