Production of toxic reactive oxygen species (ROS) due to over activity of xanthine oxidase (XO) is thought to be involved in many neuronal and vascular pathologies. A combination of xanthine (X) and XO is used as an in vitro model to produce mainly superoxide, but it has also been reported to generate hydrogen peroxide. Interestingly, it was reported that the cell death resulting from applying this combination to cultures of cerebellar granule neurons (CGNs) was prevented by catalase but not by superoxide dismutase, suggesting the predominant role of hydrogen peroxide over superoxide in this toxicity. Deferoxamine, an iron chelator that hinders the conversion of hydrogen peroxide to the highly toxic hydroxyl radical prevented the cell damage, confirming the predominant role of hydrogen peroxide and its subsequent conversion to hydroxyl radical. In the light of these findings we have examined the effect of blocking different sites on XO on cell viability in a model of X/XO toxicity in cultured CGNs to help determine which site on the enzyme is responsible for the generation of ROS (especially hydrogen peroxide). A concentration of X(100 µM)/ XO (0.02 units/ml) applied to cultured CGNs for one hour resulted in consistent and significant damage. Catalase at 300 units/ml completely prevented the damage, whereas superoxide dismutase (50-1500 units/ml) failed to provide protection. Allopurinol, an inhibitor of the molybdenum site on XO, provided significant protection at 100 µM . Diphenyleneiodonium (DPI), a known NAD(P)H-oxidase inhibitor, which can also inhibit the flavin adenine dinucleotide (FAD) site on XO (a site that is not blocked by allopurinol), also provided significant protection at 1000 nM. These results suggest that more than one site on XO is responsible for the production of toxic reactive oxygen species (especially hydrogen peroxide) and the subsequent cell death.