The modified RNA base queuine [7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine] is present in tRNA because of a unique base-exchange process catalyzed by tRNA-guanine transglycosylase (TGT). Previous studies have suggested the intermediacy of a covalent TGT-RNA complex. To exist on the reaction pathway, this covalent complex must be both chemically and kinetically competent. Chemical competence has been demonstrated by the crystal structure studies of Xie et al. [(2003) Nat. Struct. Biol. 10, 781-788]; however, evidence of kinetic competence had not yet been established. The studies reported here unequivocally demonstrate that the TGT-RNA covalent complex is kinetically capable of occurring on the TGT reaction pathway. These studies further suggest that dissociation of product RNA from the enzyme is overall rate-limiting in the steady state. Interestingly, studies comparing RNA with a 2'-deoxyriboside at the site of modification suggest a role for the 2'-hydroxyl group in stabilizing the growing negative charge on the nucleophilic aspartate (264) as the glycosidic bond to the aspartate is broken during the breakdown of the covalent complex.