Quinazolin-4(3H)-one derivatives substituted at the 6- and/or 7-position were studied as electronic probes of substrate oxidation by buttermilk xanthine oxidase. Since the enzyme active site possesses dimensional tolerance, the substituents exert an electronic effect rather than a steric effect on the catalytic parameters for oxidation. This feature permitted a Hammett plot to be made for quinazoline-oxygen substrate activity. The concave downward nature of this plot indicates that the rate-determining step for oxidation changes when electron-withdrawing substituents are placed on the substrate. This plot and kinetic isotope effects obtained with 2-deuterio derivatives of the substrates indicate the following: (i) oxidation involves nucleophile transfer to the C(2) center in concert with hydride transfer to the molybdenum center, and (ii) the formation of oxidized product is a three-step process, i.e., Michaelis complex formation, oxidation, and hydrolysis of the oxidized substrate-enzyme adduct. The role of the nucleophile in oxidation appears to be to increase the electron density in the substrate and thereby facilitate hydride transfer. The implication of this study is that similar electronic probes may be designed to study other purine-utilizing enzymes possessing a dimensionally tolerant active site.