The initial velocity of xanthine oxidase reaction catalyzed by milk xanthine oxidase is determined over the range of substrate concentrations from 1 to 0.005mM by following the consumption of molecular oxygen by a polarographic method using a Clark oxygen electrode. The Michaelis constant determined is 1.3×10^-5M. The activity is blocked by the substrate levels higher than 40μM. The constant for the substrate inhibition is calculated to be 1.3×10^-3M. The strong inhibition of the enzyme by 2-amino-4-hydroxy-6-formylpteridine develops with time: the constant for dissociation of the enzyme-inhibitor complex, K_i, is determined to be 8.0×10^-8M when the enzyme is not treated with the inhibitor prior to the start of the reaction by addition of the substrate, but 7.0×10^-9M when pretreatment for 10min is made. This potency, however, is markedly decreased by the reduction of a 7, 8-double bond or a formyl group at position 6 of the pyrazine ring of the antagonist. 2-Amino-4-hydroxy-6-formyl-7, 8-dihydropteridine and 2-amino-4-hydroxy-6-hydroxymethylpteridine inhibit the enzyme activity with the respective Ki values of 2.5×10^-5M and 10^-5M by competing with the substrate at the active center on the enzyme. These findings suggest that the strong affinity of the formylpteridine for the enzyme correlates profoundly with an oxidized structure of the pyrazine ring substituted with a formyl group at the position 6.