There has been long interest in understanding the chemical mechanisms of NAD⁺ hydrolysis. These mechanisms are characterized by pH independent and pH dependent regimes. Below pH 6 hydrolysis reaction rate is pH-independent and complete KIE analysis has determined a transition state involving an oxocarbenium ion, which has low bond orders to the nicotinamide leaving group and the incoming water nucleophile. Between pH 7 and 11 the log of the rate constant is linearly dependent on pH, and becomes pH independent at pH values above 11. This profile suggests a mechanism dependent on sugar ionization, although to date the specific mechanism by which NAD⁺ hydrolysis is accelerated by sugar ionization is unexplained. To determine the role of sugar ionization on hydrolysis, we investigated the transition state structure of NAD⁺ hydrolysis at pH 9 using competitive kinetic isotope effects and density functional calculations (Gaussian03, ISOEFF07). A family of KIEs was determined for the hydrolysis reaction at pH 9, using competitive-radiolabel method. Primary isotope effects were 1.15 for 1'_N-C¹⁴-NAD⁺ and 1.024 for 1_N-N¹⁵-NAD⁺. Secondary KIEs were 1.22 for 1'_N-H³- and 1.09 for 2'_N-H³-NAD⁺, respectively. The KIEs suggest strong nucleophile participation at the transition state in the hydrolysis reaction. However, vibrationally less constrained environments for both the 1'_N- and 2'_N-hydrogens suggest that sugar ionization leads to an epoxide-product like transition state.