Xanthine oxidoreductase (XOR) is a mammalian enzyme that possesses a series of redox centers, which use either NAD⁺ or molecular oxygen for oxidation of the purines xanthine and hypoxanthine to uric acid. The ability of XOR to act as an NADH oxidase is a less well recognized function of the enzyme, and it is this function that we used to explore the metabolism of glyceryl trinitrate. The antiplatelet effect of nitric oxide (NO) on platelet aggregation was used as a bioassay to assess the bioconversion of glyceryl trinitrate to NO by XOR. The thromboxane mimetic U46619, 2 μM, was used to stimulate platelet aggregation in platelet-rich plasma prepared from healthy drug-free human volunteers. All incubations were carried out at 37°C for 2 min after the addition of U46619. XOR produced a dose-dependent antiaggregant effect when incubated with glyceryl trinitrate (GTN), 220 μM. This did not occur when GTN or XOR was incubated with platelet-rich plasma independently. The antiaggregant effect of XOR plus GTN was dose dependently inhibited by allopurinol, with an IC₅₀ of 100 μM. The addition of superoxide dismutase (SOD), 100 U/ml produced a shift to the left in the antiaggregant dose-response curve for XOR. The IC₅₀ for XOR at 200 U/l without SOD was decreased to 80 U/l with SOD. Oxyhemoglobin, an extracellular NO scavenger, produced a dose-dependent, noncompetitive inhibition of the antiaggregant effect of XOR plus GTN. These findings suggest that GTN may be reduced to NO in vitro by the enzyme XOR in sufficient amounts to inhibit platelet aggregation.