The kinetics of the reaction of milk xanthine oxidase (XO) with o-phenanthroline and 1,7-dimethylxanthine are investigated by optical and circular dichroism (CD) spectroscopic techniques. o-Phenanthroline and 1,7-dimethylxanthine are observed to react with XO, resulting in a slow decrease in the absorbance and CD signals of the enzyme. The results suggest the existence of a multi-binding mechanism in the reaction of XO with o-phenanthroline with two molecules of the substrate binding to one molecule of the enzyme. However, 1,7-dimethylxanthine binds reversibly with the enzyme with 1∶1 stoichiometry. The kinetic effect experienced individually by the flavin adenine dinucleotide (FAD) chromophore of XO has been separately resolved, and shows that at least one molecule of o-phenanthroline may bind near the FAD centre of the enzyme. 1,7-Dimethylxanthine is expected to bind to the molybdenum centre in analogy with xanthine.