The transition state of nucleoside hydrolase from the trypanosome Crithidia fasciculata has been characterized by multiple Vmax/Km kinetic isotope effects with labeled inosine and adenosine as substrates. Nucleoside hydrolase catalyzes the hydrolysis of the N-glycosidic linkage of the commonly occurring purine and pyrimidine nucleosides, with Vmax/Km ranging over 2 orders of magnitude. The kinetic isotope effects for inosine were [1'-3H] = 1.150 +/- 0.006, [2'-3H] = 1.161 +/- 0.003, [1'-14C] = 1.044 +/- 0.004, [9-15N] = 1.026 +/- 0.004, [4'-3H] = 0.992 +/- 0.003, and [5'-3H] = 1.051 +/- 0.003. The magnitude of the kinetic isotope effects for inosine, an equivalent [1'-3H] kinetic isotope effect for the poor substrate adenosine, and the rapid equilibrium random kinetic mechanism [Parkin D, W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., & Schramm, V. L. (1991) J. Biol. Chem. (in press)] all indicate that the isotope effects are fully expressed. The kinetic and solvent deuterium isotope effects have been used to analyze the transition-state structure using bond energy bond order vibrational analysis. The transition state involves a protonated hypoxanthine leaving group with a C-N glycosidic bond elongated to approximately 2 A. The ribose group contains substantial carbocationic character, unusually strong hyperconjugation of H2', and a bond length of approximately 3 A to the incoming oxygen nucleophile. The remote isotope effect (4'-3H and 5'-3H) and the results of transition-state calculations provide the most detailed description of the steric and bonding properties of an enzyme-stabilized transition state.