Pertussis toxin from Bordatella pertussis catalyzes the ADP ribosylation of several G-proteins, using NAD+ as a substrate. In the absence of an acceptor protein, the toxin acts as a NAD+ glycohydrolase. Pertussis toxin is one of the virulent factors for whooping cough and therefore a target for site-specific inhibitors based on the transition state structure. A family of kinetic isotope effects was determined for the hydrolysis reaction, using NAD+ labeled with 3H, 14C, and 15N as substrates. Primary isotope effects were 1.021 +/- 0.001 for [1'N-14C]NAD+ and 1.021 +/- 0.004 for [1N-15N]NAD+, and the double-primary effect of [1'N-14C,1N-15N]NAD+ was 1.049 +/- 0.004. Secondary kinetic isotope effects were 1.207 +/- 0.010 for the [1'N-3H]-, 1.144 +/- 0.005 for the [2'N-3H]-, 0.989 +/- 0.001 for the [4'N-3H]-, and 1.019 +/- 0.004 for the [5'N-3H]NAD+, respectively. Commitment to catalysis was excluded by isotope trapping experiments, and the experimental kinetic isotope effects were independent of pH. The measured isotope effects are therefore intrinsic. The isotope effects are remarkable because they indicate an oxocarbenium-like ribose ring at the transition state but a stiffer than expected vibrational environment for C1' at the reaction center. On the basis of these isotope effects, a bond order vibrational analysis was performed to locate a transition state structure consistent with the isotope effects. The kinetic isotope effects predict a residual bond order to the nicotinamide leaving group of 0.11, corresponding to a distance of 2.14 A. Participation of the water nucleophile is weak, consistent either with an S(N)1-like transition state with no water interaction or with the water oxygen no closer than 3.5 A from the reaction center. The positive charge of the ribose oxocarbenium is stabilized by delocalization between the C1'-O4' and C1'-C2' bonds. The enzyme contacts restrict the vibrational environment of the reaction coordinate requiring increased bonding force constants for the enzyme-stabilized transition state. NAD+ analogues with the nicotinamide ribose replaced by an iminoribitol ring, mimicking the flattened ribose ring of the transition state, are expected to be transition state inhibitors.