A useful property of DMSO solvent has been exploited to reveal a new catalytic route for cytidine amino proton exchange, relevant to exchange in the macromolecular state, but hidden in aqueous solution. Additional exchange mechanisms in aqueous monomeric cytidine (and adenosine) are obscured by the formation of a fast-exchanging endocyclic-protonated intermediate, which dominates the kinetics. Endocyclic nucleobase protonation could be circumvented in the presence of buffer conjugate acid by the use of DMSO/water solvent, permitting the first unequivocal observation buffer acid-catalyzed exchange from the neutral, unprotonated nucleobase, i.e., general acid catalysis. Because buffer ionization is greatly reduced in DMSO through anion desolvation, nucleobase protonation is suppressed in the presence of buffer acid. Evidence is presented to describe this catalytic route as one involving hydrogen bond formation between the buffer acid and the endocyclic protonation site, C(N-3). Since this same configuration is found in Watson-Crick hydrogen bonding, experiments are presented to demonstrate faster cytidine amino proton exchange with the formation of the G-C base pair in DMSO. The importance of this mechanism in past aqueous monomer studies and in the interpretation of macromolecular (DNA) hydrogen exchange is discussed.