The transition state structure has been determined for the pH-independent solvolytic hydrolysis of NAD(+). The structure is based on kinetic isotope effects (KIEs) measured for NAD(+)'s labeled in various positions of the ribose ring and in the leaving group nitrogen. The KIEs for reactions performed at 100 degrees C in 50 mM NaOAc (pH 4.0) were as follows: 1-N-15, 1.020 +/- 0.007; 1'-C-14, 1.016 +/- 0.002; [1-N-15,1'-C-14], 1.034 +/- 0.002; 1'-H-3, 1.194 +/- 0.005; 2'-H-3, 1.114 +/-0.004; 4'-H-3, 0.997 +/- 0.001; 5'-H-3, 1.000 +/- 0.003; 4'-O-18, 0.988 +/- 0.007. The transition state structure was determined using bond energy/bond order vibrational analysis to predict KIEs for trial transition; state models. The structure that most closely matches the experimental KIEs defines the transition state. A structure interpolation method was developed to generate trial transition state structures and thereby systematically search reaction coordinate space. Structures are generated by interpolation between reference structures, reactant NAD(+) and a hypothetical {ribo-oxocarbenium ion plus nicotinamide} structure. The point in reaction coordinate space where all the predicted KIEs matched the measured ones was considered to locate the transition state structure. This occurred when the residual bond order to the leaving group nicotinamide, n(LG,TS), was 0.02 (bond length = 2.65 Angstrom) and the bond order to the approaching nucleophile, n(Nu,TS), was 0.005 (3.00 Angstrom). Thus, bond-breaking and bond-making in this A(N)D(N) reaction are asynchronous, and the transition state has a highly oxocarbenium ion-like character.