Diphtheria toxin (DTA) uses NAD(+) as an ADP-ribose donor to catalyze the ADP-ribosylation of eukaryotic elongation factor 2. This inhibits protein biosynthesis and ultimately leads to cell death. In the absence of its physiological acceptor, DTA catalyzes the slow hydrolysis of NAD(+) to ADP-ribose and nicotinamide, a reaction that can be exploited to measure kinetic isotope effects (KIEs) of isotopically labeled NAD(+)s. Competitive KIEs were measured by the radiolabel method for NAD(+) molecules labeled at the following positions: 1-N-15 = 1.030 +/- 0.004, 1'-C-14 = 1.034 +/- 0.004, (1-N-15,1'-C-14) = 1.062 +/- 0.010, 1'-H-3 = 1.200 +/- 0.005, 2'-H-3 = 1.142 +/-0.005, 4'-H-3 = 0.990 +/- 0.002, 5'-H-3 = 1.032 +/- 0.004, 4'-O-18 = 0.986 +/- 0.003. The ring oxygen, 4'-O-18, KIE was also measured by whole molecule mass spectrometry (0.991 +/- 0.003) and found to be within experimental error of that measured by the radiolabel technique, giving an overall average of 0.988 +/- 0.003. The transition state structure of NAD(+) hydrolysis was determined using a structure interpolation method to generate trial transition state structures and bond-energy/bond-order vibrational analysis to predict the KIEs of the trial structures. The predicted KIEs matched the experimental ones for a concerted, highly oxocarbenium ion-like transition state. The residual bond order to the leaving group was 0.02 (bond length = 2.65 Angstrom), while the bond order to the approaching nucleophile was 0.03 (2.46 Angstrom). This is an A(N)D(N) mechanism, with both leaving group and nucleophilic participation in the reaction coordinate. Fitting the transition state structure into the active site cleft of the X-ray crystallographic structure of DTA highlighted the mechanisms of enzymatic stabilization of the transition state. Desolvation of the nicotinamide ring, stabilization of the oxocarbenium ion by apposition of the side chain carboxylate of Glu148 with the anomeric carbon of the ribosyl moiety, and the placement of the substrate phosphate near the positively charged side chain of His21 are all consistent with the transition state features from KIE analysis.