In the previous papers, it has been shown that the substrate inhibition of xanthine oxidase (xanthine: O2 oxidoreductase, EC 1. 2. 3. 2) induced by excess purines requires a small amount of exogenous metallic ions. Among these ions, Cu²+ was the most typical one. At any stage of enzyme reaction, the inhibition began immediately on addition of a small amount of Cu²+ such as 6.6 X 10-7 M. Since the depressed activity was not restored by the addition of chelating agents such as histamine and EDTA, it was suggested that the substrate, Cu²+ and enzyme form a stable inactive enzyme complex, from which chelating agent can no longer remove Cu. The present communication describes the further investigations concerned with the formation of the substrate-enzyme complex in the presence of Cu²+ and with the catalytic nature of this complex on other substrate and acceptor systems.