Abstract A spin-labeled adenine derivative [N 6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine; SLAD] is found to be a very slow substrate of xanthine oxidase based on the observed reduction of enzyme by SLAD under anaerobic conditions. A room-temperature EPR spectrum of SLAD in the presence of oxidized xanthine oxidase shows the appearance of “wings” on the three-line spectrum of the free spin-label, indicating formation of an E·SLAD complex. This spectrum can be obtained on a timescale that is short compared to catalysis. Using this spectral change as an experimental probe, the room-temperature K d's of SLAD binding to oxidized xanthine oxidase at various pH's have been determined. Obtained K d values are 1.5 ± 0.3 mM, 1.6 ± 0.3 mM, and 1.5 ± 0.3 mM at pH 10.0, 8.5, and 7.0, respectively, indicating no significant difference in the equilibrium dissociation of SLAD from enzyme upon pH change. These results are consistent with the calculated equilibrium dissociation constant for substrate binding to oxidized molybdenum center based on K d to reduced enzyme and the perturbation of Mo VI/Mo V and Mo V/Mo IV reduction potentials by product and substrate analogs.