Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with betaine aldehyde as an intermediate. In this study, primary deuterium and solvent kinetic isotope effects have been used to elucidate the mechanism for substrate oxidation by choline oxidase using both steady-state kinetics and rapid kinetics techniques. The (D)(k(cat)/K-m) value with 1,2-[H-2(4)]-choline at saturating oxygen concentration was independent of pH in the range between 6.5 and 10, with a value of similar to10.6, indicating that CH bond cleavage is not masked by other titratable kinetic steps belonging to the reductive half-reaction. In agreement with this conclusion, a D ked value of similar to8.9 was determined at pH 10 for the anaerobic reduction of the flavin by choline, irrespective of whether aqueous or deuterated solvent was used. At pH 10, both the (D)(O)(2)(k(cat)/K-m) and the (D)(O)(2)k(red) values were not different from unity with choline or 1,2-[H-2(4)]-choline, while the (D)k(cat) and (D)(O)(2)k(cat) values were 7.3 and 1.1, respectively. The k(cat) and k(red) values were 133 s(-1) and 135 s(-1) with betaine aldehyde and 60 s(-1) and 93 s(-1) with choline. These data are consistent with a chemical mechanism in which the choline hydroxyl proton is not in flight in the transition state for CH bond cleavage and with chemical steps of flavin reduction by choline and betaine aldehyde being rate limiting for the overall turnover of the enzyme.