Ricin toxin A-chain (RTA) depurinates 28 S ribosomal RNA and small stem-loop RNAs at the first adenosine residue in a 5‘-GAGA-3‘ tetraloop. The transition state for depurination of stem-loop RNA by RTA was determined from kinetic isotope effects (KIEs). A stem-loop RNA, called A-10 (5‘-GGCGAGAGCC-3‘), was synthesized using isotopically labeled ATP. KIEs were measured for RNA substrates with adenylates containing [1‘-14C], [9-15N], [1‘-14C,9-15N], [7-15N], [1‘-3H], [2‘-3H], [4‘-3H], or [5‘-3H]. Substrate-trapping experiments established that the Michaelis complex of RTA·[14C]A-10 dissociates to free enzyme and [14C]A-10 at least 20 times more frequently than its conversion to products, establishing minimal forward commitment to catalysis. KIEs were used to interpret the transition-state structure. The experimental KIEs differ from previous N-ribohydrolase chemistries. Large KIEs were measured for [1‘-3H] (1.163 ± 0.009) and [7-15N] (0.981 ± 0.008). A modest isotope effect occurred with [9-15N] (1.016 ± 0.005), and small KIEs were observed with [1‘-14C] (0.993 ± 0.004) and [2‘-3H] (1.012 ± 0.005). The experimental KIEs were analyzed using bond vibrational and quantum chemical approaches, which demonstrated that a complex is formed of RTA with the RNA ribooxocarbenium ion and adenine that is in equilibrium with the Michaelis complex. A slow, irreversible, and nonchemical step is followed by nucleophilic attack by water. Release of the depurinated A-10 and adenine products is rapid. Other N-ribohydrolases catalyze dissociative concerted ANDN (SN2) transition states with weak participation of the leaving group and nucleophile. The KIEs for RTA establish a stepwise DN*AN mechanism and the existence of a cationic intermediate with a finite lifetime. The conformation of the ribosyl ring in the enzyme-stabilized RNA·ribooxocarbenium ion is 3‘-endo, with an unusual dihedral angle of approximately 50° between C2‘-H2‘ and the vacant p-orbital of atom C1‘. This conformation, which is unprecedented in N-ribohydrolases, is consistent with the geometry imposed by the stem-loop RNA backbone. These results establish that transition state analysis based on KIEs can be extended to the reactions of nucleic acid chemistry.